U.S. patent application number 11/024053 was filed with the patent office on 2006-01-05 for dry powder cells and cell culture reagents and methods of production thereof.
Invention is credited to Christine M. Biddle, Jeffrey W. Biddle, Laurel Biddle, William C. Biddle, Barbara M. Dadey, Richard Fike, Richard F. Hassett, William Whitford.
Application Number | 20060003448 11/024053 |
Document ID | / |
Family ID | 34748847 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003448 |
Kind Code |
A1 |
Fike; Richard ; et
al. |
January 5, 2006 |
Dry powder cells and cell culture reagents and methods of
production thereof
Abstract
The present invention relates generally to nutritive (e.g., cell
culture) medium, medium supplement, media subgroup and buffer
formulations. Specifically, the present invention provides powdered
nutritive medium, medium supplement and medium subgroup
formulations, particularly cell culture medium supplements
(including powdered sera such as powdered fetal bovine serum
(FBS)), medium subgroup formulations and cell culture media
comprising necessary nutritive factors that facilitate the in vitro
cultivation of cells. The invention further provides such powdered
formulations that produce particular or desired final ionic and/or
pH conditions upon reconstitution with a solvent. The invention is
also directed to methods of production of these formulations, and
also provides kits and methods for cultivation of prokaryotic and
eukaryotic cells using these formulations. The invention also
relates to methods of producing sterile formulations, and to
methods for producing dry cell powders. The invention also provides
cell, media, media supplement, media subgroup and buffer powders
produced by the methods of the invention.
Inventors: |
Fike; Richard; (Clarence,
NY) ; Whitford; William; (Santa Fe, NM) ;
Biddle; William C.; (Buffalo, NY) ; Biddle;
Laurel; (Buffalo, NY) ; Biddle; Christine M.;
(Buffalo, NY) ; Biddle; Jeffrey W.; (Buffalo,
NY) ; Hassett; Richard F.; (Tonewanda, NY) ;
Dadey; Barbara M.; (East Aurora, NY) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Family ID: |
34748847 |
Appl. No.: |
11/024053 |
Filed: |
December 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60533055 |
Dec 30, 2003 |
|
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Current U.S.
Class: |
435/404 |
Current CPC
Class: |
C12N 5/0018
20130101 |
Class at
Publication: |
435/404 |
International
Class: |
C12N 5/02 20060101
C12N005/02 |
Claims
1. A dry format media supplement, said supplement comprising at
least two powder components and said supplement reconstitutable for
addition to a cell culture medium, said medium capable of
supporting cell growth and/or expansion and/or production
biomolecules.
2. The dry media supplement according to claim 1 comprising a
mixture of at least one first component selected from the group
consisting of an amino acid or salt thereof, a polysaccharide, a
solubilizing agent, a vitamin, a lipid, a fatty acid, a hormone, a
growth factor, a differentiation factor, an active polypeptide, an
iron chelator, a divalent metal salt, a carbon source, a monovalent
metal salt, a pH buffer, a polyanion, a polycation, a surfactant,
an antioxidant, a trace element salt, a nucleotide, a heterocyclic
base and a nucleoside; and at least one second component having a
different chemical constitution than said first component, said
second component selected from the group consisting of an amino
acid or salt thereof, a polysaccharide, a solubilizing agent, a
vitamin, a lipid, a fatty acid, a hormone, a growth factor, a
differentiation factor, an active polypeptide, an iron chelator, a
divalent metal salt, a carbon source, a monovalent metal salt, a pH
buffer, a polyanion, a polycation, a surfactant, an antioxidant, a
trace element salt, a nucleotide, a heterocyclic base and a
nucleoside.
3. The supplement according to claim 2 wherein said supplement
comprises at least one amino acid or salt thereof selected from the
group consisting of alanine, cysteine, cystine, aspartic acid,
glutaminc acid, phenylalanine, glycine, histidine, isoleucine,
lysine, leucine, methionine, asparagine, proline, hydroxyproline,
glutamine, arginine, serine, threonine, valine, tryptophan and
tyrosine.
4. The supplement according to claim 1 comprising at least one
component selected from the group consisting of alanine, cysteine,
cystine, aspartic acid, glutaminc acid, phenylalanine, glycine,
histidine, isoleucine, lysine, leucine, methionine, asparagine,
proline, hydroxyproline, glutamine, arginine, serine, threonine,
valine, tryptophan and tyrosine, in free or salt form.
5. The supplement according to claim 1 reconstituted in a polar
solvent.
6. The supplement according to claim 1 reconstituted in essentially
physiologic pH water.
7. The supplement according to claim 2 comprising a combination of
at least 3 amino acids or any salt thereof.
8. The supplement according to claim 4 further comprising at least
one vitamin.
9. The supplement according to claim 1 wherein the powder is
prepared by at least one method selected from the group consisting
of milling, impacting, extruding and cutting or breaking, wet
granulation, high shear granulation, pan granulation and fluidized
bed agglomeration.
10. The milled supplement according to claim 9 wherein the milling
apparatus is selected from the group consisting of ball mill, roll
mill, fitz mill, comill, jet mill and hammer mill, and any other
mechanical particle size attrition device.
11. The supplement according to claim 2 comprising at least two
components selected from the group consisting of an amino acid or
salt thereof, a vitamin, and a carbon source.
12. The supplement according to claim 2 comprising at least two
components selected from the group consisting of a metal chelator,
lipids, fatty acids, a divalent metal salt, a pH buffer, and a
carbon source.
13. The supplement according to claim 2 comprising at least two
components selected from the group consisting of a polyanion, a
polycation, a surfactant, an antioxidant, a carbon source, a trace
metal element salt, a nucleotide, a heterocyclic base and a
nucleoside.
14. A method of reconstituting a dry medium supplement powder
comprising: obtaining a powder according to claim 1; and dissolving
said supplement in essentially neutral pH water or polar
solvent.
15. A dry powder medium supplement for supplementing cells growing
in culture, the improvement being formulating said supplement
powder to reconstitute in essentially neutral pH water or polar
solvent.
16. A dry powder medium supplement according to claim 15 for
supplementing cells growing in culture, the improvement being pH
adjustment after reconstitution is not required.
17. A method of culturing cells comprising: growing cells in
culture for at least 2 days to form a mature culture;
reconstituting a supplement powder using essentially neutral pH
water and or polar solvent to form a reconstituted supplement; and
adding said reconstituted supplement to said mature culture.
18. The method according to claim 17 wherein growth expression
and/or production of said cells in culture is increased from that
of the mature culture.
19. The supplement according to claim 1 in kit form comprising at
least two containers of different composition of components wherein
at least one container contains a mixture of at least two
components.
20. The supplement according to claim 19 where reconstitution of
the components of at least one container is in a polar solvent and
the reconstitution of the components of at least a second container
is in essentially physiologic pH water.
21. The supplement according to claim 2 wherein said mixture is a
homogenous mixture.
22. The method according to claim 17 wherein said adding comprises
delivery by using a pressure feed wherein said pressure is a result
of gravity, pneumatic displacement, fluid displacement, or other
physical displacement.
23. The method according to claim 22 wherein the pressure feed is
provided by a pump selected from the group consisting radial flow,
rotary, axial flow, regenerative, turbine, plunger, diaphragm, cam
and piston, peristaltic, gear, lobe, piston, screw, syringe,
metering, sliding-vane, hydraulic, jet, volumetric displacement,
and other reciprocating or positive displacement pumps.
24. The method according to claim 17, wherein adding is achieved at
a ratio selected from the group consisting of about 1%/day to about
500%/day.
25. The method according to claim 17 wherein the adding is metered
according to a feed back circuit based on at least one measured
concentration or size from a component in the medium.
26. The supplement of claim 2 wherein the active polypeptide is
selected from the group consisting of an enzyme, a transport
protein, a membrane stabilizer, a neurotransmitter, a
differentiating agent and a binding or sequestering agent.
27. The medium supplement powder according to claim 8 wherein the
vitamin is selected from the group consisting of ascorbate or
ascorbic acid or salt thereof, biotin, pantothenate or pantothenic
acid, bitartrate, choline chloride, cyanocobalamin, D- or DL-alpha
tocopherol or tocopherol acetate, folic acid, folinic acid or a
salt thereof, i-inositol, carnitine, lipoic acid, linoleic acid,
menadione or salt thereof, niacinamide, nicotinic acid,
para-aminobenzoic acid, pyridoxal 5-phosphate, pyidoxal HCl,
pyridoxamine mono or di hydrochloride, retinoic acid, riboflavin,
riboflavin-5-phosphate sodium dihydrate, thiamine HCl, Thiamine
monophosphate, vitamin A or salt thereof, Vitamin D2 and vitamin
D3.
28. The supplement according to claim 1 wherein the at least two
powder components are milled to produce a mean diameter ratio of a
largest component to a smallest component of 1:1 to 10:1.
29. The supplement according to claim 1 wherein the at least two
powder components are blended using tumble blender including but
not limited to drum blender, V-blender, ribbon blender, cone
blender, slant-cone blender and double-cone blender sufficient
mixing time and speed to attain a homogenous mixture of
components.
30. The supplement according to claim 1 wherein the at least two
powder components are blended to homogeneity.
31. A method of producing a medium supplement, said method
comprising: obtaining a dry format media supplement said supplement
comprising at least two powder components and said supplement
reconstitutable for addition to a cell culture medium, said medium
capable of supporting cell growth, biomolecule production and/or
expansion; and dissolving said supplement in a solvent to produce
said medium supplement; and optionally adding said medium
supplement to said medium.
32. The method according to claim 31, wherein at least one of said
at least two components is present at a concentration in excess of
a 1.times. concentration with respect to the medium being
supplemented.
33. The method according to claim 32, wherein at least one of said
at least two components is present at a concentration of from
greater than a 1.times. to about a 5.times., 10.times., 20.times.,
50.times., 75.times., 100.times., 120.times., 150.times.,
200.times., 250.times., 500.times., 750.times. or up to a
1000.times. concentration with respect to the medium being
supplemented.
34. The method according to claim 31, wherein at least one of said
at least two components is present at a concentration in excess of
about 4.times. concentration with respect to the medium being
supplemented.
35. The method according to claim 31, wherein at least one of said
at least two components is present at a concentration in excess of
about a 9.times. concentration with respect to the medium being
supplemented.
36. The method according to claim 34, wherein at least one of said
at least two components is present at a concentration of about
5.times. with respect to the medium being supplemented.
37. The method according to claim 35, wherein at least one of said
at least two components is present at a concentration of about
10.times. with respect to the medium being supplemented.
38. The supplement according to claim 1 wherein a first component
of the medium being supplemented is omitted or is present at a
concentration with respect to said medium being supplemented in
comparison to a second component with respect to said medium being
supplemented at a ratio greater than 0, range 10-6 to 0.9.
39. The supplement according to claim 38 wherein said first
component is selected from the group consisting of alanine,
cysteine, cystine, aspartic acid, glutaminc acid, phenylalanine,
glycine, histidine, isoleucine, lysine, leucine, methionine,
asparagine, proline, hydroxyproline, glutamine, arginine, serine,
threonine, valine, tryptophan and tyrosine, an alkali metal salt, a
pH buffer and a surfactant.
40. The supplement according to claim 1 wherein all components are
soluble in essentially physiologic pH water at a concentration of
from about 2.times., about 4.times., about 5.times., about
10.times., about 15.times., about 20.times., about 25.times., about
30.times., about 40.times., about 45.times., to about 48.times. or
about 50.times. or from about 50.times. to about 100.times., about
120.times., about 150.times., about 180.times., about 190.times. or
about 200.times. of the medium being supplemented.
41. The supplement according to claim 1 wherein all components are
of non-animal origin.
42. The supplement according to claim 1 wherein said supplement is
chemically defined.
43. A supplement according to claim 1 wherein no component is serum
or a product of serum.
44. The supplement according to claim 5 comprising alanine,
cysteine, cystine, aspartic acid, glutaminc acid, phenylalanine,
glycine, histidine, isoleucine, lysine, leucine, methionine,
asparagine, proline, hydroxyproline, glutamine, arginine, serine,
threonine, valine, tryptophan and tyrosine, each individually in
free or salt form. Discuss phosphate technology--either this or
separate patent appl. Or trade secret
45. The reconstituted supplement according to claim 5 comprising
alanine, cysteine, cystine, aspartic acid, glutaminc acid,
phenylalanine, glycine, histidine, isoleucine, lysine, leucine,
methionine, asparagine, proline, hydroxyproline, glutamine,
arginine, serine, threonine, valine, tryptophan and tyrosine, each
individually at a concentration between about 2.times. to about
50.times..
46. The reconstituted supplement according to claim 6 comprising
alanine, cysteine, cystine, aspartic acid, glutaminc acid,
phenylalanine, glycine, histidine, isoleucine, lysine, leucine,
methionine, asparagine, proline, hydroxyproline, glutamine,
arginine, serine, threonine, valine, tryptophan and tyrosine, each
individually at a concentration between about 2.times. to about
50.times..
47. The supplement according to claim 9 wherein the powder is
prepared by fluidized bed agglomeration.
48. The supplement according to claim 9 comprising a sugar.
49. The supplement according to claim 9 comprising a polymeric
binder such as methyl cellulose and deriviative of starch, ethyl
cellulose and polyvinylpyttolidone (PVP).
50. The dry format media supplement, according to claim 1, wherein
at least a first powder component is designated as 1.times. per
unit weight based on the medium to be supplemented and a second
powder component is greater than 1.4.times. or less than 0.8.times.
per said unit weight.
51. The supplement according to claim 50 wherein at least said
second powder component is not present in said medium to be
supplemented.
52. The supplement according to claim 1 that upon reconstitution
with a solvent results in at least one component concentration
greater than about 2.times. that of said medium component
concentration.
53. The supplement according to claim 1 wherein at least one
component is soluble at at least 2.times. in physiologic pH
water.
54. The supplement according to claim 1 comprising bicarbonate.
55. The supplement according to claim 54 wherein the bicarbonate is
a salt of a monovalent metal.
56. The supplement according to claim 15 comprising
bicarbonate.
57. The supplement according to claim 56 wherein the bicarbonate is
a salt of a monovalent metal.
58. The supplement according to claim 1 comprising trace element
salts.
59. The supplement according to claim 60 wherein said trace
elements salts are homogenously distributed throughout the dry
format media supplement.
60. The method according to claim 17 wherein said culturing results
in creation of or an increased production of a desired cell
component, product or function.
61. The method according to claim 25 wherein the at least one
measured concentration or size comprises at least one selected from
the group consisting of cell volume, number, size, concentration of
glucose, any amino acid, a metabolic product or metabolite, an
active oxygen, an active oxygen product, lactate, pH, Na, K, Ca,
Se, viscosity, light absorbance, color change and protein.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/533,055, filed Dec. 30, 2003, the
disclosure of which is entirely incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to cells, nutritive
media, media supplements, media subgroups and buffer formulations.
Specifically, the present invention provides dry powder nutritive
medium formulations, particularly cell culture medium formulations,
comprising all of the necessary nutritive factors that facilitate
the in vitro cultivation of cells, and methods and means of
supplementing of these media formulations. The invention also
specifically relates to methods of producing dry powder media
supplements, such as dry powder sera (e.g., fetal bovine serum),
dry powder nutrient supplements, concentrated supplements and
methods of making and using same. The invention also relates to dry
powder media, media supplement, media subgroup and buffer
formulations that produce particular ionic and pH conditions upon
rehydration, without the need for adjustment of such conditions
prior to use.
[0004] The invention also relates to methods of preparing sterile
dry powder nutritive media, media supplements (particularly dry
powder sera amino acid containing powders and carbon source, e.g.,
sugar containing powders), media subgroups and buffer formulations.
The invention also relates to dry powder nutritive media, media
supplements, media subgroups, buffer formulations and cells
prepared by these methods. The invention also relates to kits and
methods for cultivation of prokaryotic and eukaryotic cells using
these dry powder nutritive media, media supplements, media
subgroups and buffer formulations.
[0005] 2. Related Art
[0006] Cell Culture Media
[0007] Cell culture media provide the nutrients necessary to
maintain and grow cells in a controlled, artificial and in vitro
environment. Characteristics and compositions of the cell culture
media vary depending on the particular cellular requirements.
Important parameters include osmolality, pH, and nutrient
formulations.
[0008] Media formulations have been used to cultivate a number of
cell types including animal, plant and bacterial cells. Some cells
are capable of growing on a solid or semi-solid medium, but cells
derived from other than the simplest life forms generally are
cultured in liquid phase. Cells cultivated in culture media
catabolize available nutrients and produce useful biological
substances such as monoclonal antibodies, hormones, growth factors,
viruses and the like. Such products have therapeutic applications
and, with the advent of recombinant DNA technology, cells can be
engineered to produce large quantities of these products. Thus, the
ability to cultivate cells in vitro is not only important for the
study of cell physiology, but is also necessary for the production
of useful substances which may not otherwise be obtained by
cost-effective means.
[0009] As the cells catabolize nutrients the environment in which
the cells grow is constantly being altered. Catabolic products may
remain in culture or may require the cultured cells to catabolize
these also to maintain cell health. The medium is thus constantly
changing. The requirements of the cultured cells may be changing
also. Especially for media optimized for a particular cell type or
especially a particular production task as the cells grow (and
produce) the medium becomes less conducive to the desired result.
Supplementation of medium has been effectively used to prolong
culture or to maintain or improve production. Several
supplementation programs have been used. For example, a single
bolus or multiple boli have been added to culture to replenish or
sometimes modify medium constituents. Continuous feed programs have
also been tried. Supplementation of the growing culture can
maintain growth and productivity of the cultured cells over
extended time periods.
[0010] Simple supplementation might entail adding original medium
to provide the same nutrients, but at a different final
concentration (as diluted by the partially spent medium). However,
since not all media components, e.g., sodium and chloride, are
altered, preferably supplements will comprise a set of ingredients
less than that of the original medium. Although some ingredients
are preferably omitted, a supplement might contain ingredients not
present in the original medium being supplemented. Concentrations
are preferably adjusted to take into account the different rates of
catabolism of different components, to bring about, e.g., induce, a
desired change in cell metabolism, to ameliorate the buildup of
undesired, for example, toxic, products and/or for manufacturing or
stability concerns. Preferred supplements may contain some
ingredients in concentrated form; that is at a concentration
greater than found in the original medium being supplemented. For
example, one or more ingredients of the supplement may be present
at a concentration about or exceeding 1.5, 2, 3, 4, 5, 7, 8, 10,
12, 15, 20, 25, 30, 40, 50, 60, 70, 75, 100, 125, 150, 200 times or
even greater that of the medium being supplemented; while other
ingredients may be present at a concentration the same as or about
(.+-.10%) the same as the concentration in the medium being
supplemented. Preferably one or several ingredients are omitted
from the supplement. In some embodiments multiple supplements are
used, e.g., for separate storage conditions, to allow for the
culturist's special choices, for induction, for different stages of
culture, to allow more precise control of individual ingredients or
a set of ingredients, and/or to prevent the concentration of of one
or more ingredients from exceeding that desired.
[0011] Cell culture media formulations have been well documented in
the literature and a number of media are commercially available. In
early cell culture work, media formulations were based upon the
chemical composition and physicochemical properties (e.g.,
osmolality, pH, etc.) of blood and were referred to as
"physiological solutions" (Ringer, S., J. Physiol.. 3:380-393
(1880); Waymouth, C., In: Cells and Tissues in Culture, Vol. 1,
Academic Press, London, pp. 99-142 (1965);Waymouth, C., In Vitro
6:109-127 (1970)). However, cells in different tissues of the
mammalian body are exposed to different microenvironments with
respect to oxygen/carbon dioxide partial pressure and
concentrations of nutrients, vitamins, and trace elements;
accordingly, successful in vitro culture of different cell types
will often require the use of different media formulations. Typical
components of cell culture media include amino acids, organic and
inorganic salts, vitamins, trace metals, sugars, lipids and nucleic
acids, the types and amounts of which may vary depending upon the
particular requirements of a given cell or tissue type. Often,
particularly in complex media compositions, stability problems
result in toxic products and/or lower effective concentrations of
required nutrients, thereby limiting the functional life-span of
the culture media. For instance, glutamine is a constituent of
almost all media that are used in culturing of mammalian cells in
vitro. Glutamine decomposes spontaneously into pyrolidone
carboxylic acid and ammonia. The rate of degradation can be
influenced by pH and ionic conditions but in cell culture media,
formation of these breakdown products often cannot be avoided
(Tritsch et al., Exp. Cell Res. 28:360-364(1962)).
[0012] Wang et al. (In Vitro 14(8):715-722 (1978)) have shown that
photoproducts such as hydrogen peroxide, which are lethal to cells,
are produced in Dulbecco's Modified Eagle's Medium (DMEM).
Riboflavin and tryptophan or tyrosine are components necessary for
formation of hydrogen peroxide during light exposure. Since most
mammalian culture media contain riboflavin, tyrosine and
tryptophan, toxic photoproducts are likely produced in most cell
culture media.
[0013] To avoid these problems, researchers make media on an "as
needed" basis, and avoid long term storage of the culture media.
Commercially available media, typically in dry power form, serves
as a convenient alternative to making the media from scratch, i.e.,
adding each nutrient individually, and also avoids some of the
stability problems associated with liquid media. However, only a
limited number of commercial culture media are available, except
for those custom formulations supplied by the manufacturer.
[0014] Although dry powder media formulations may increase the
shelf-life of some media, there are a number of problems associated
with dry powdered media, especially in large scale application.
Production of large media volumes requires storage facilities for
the dry powder media, not to mention the specialized media kitchens
necessary to mix and weigh the nutrient components. Due to the
corrosive nature of dry powder media, mixing tanks must be
periodically replaced.
[0015] Typically, cell culture media formulations are supplemented
with a range of additives, including undefined components such as
fetal bovine serum (FBS) (10-20% v/v) or extracts from animal
embryos, organs or glands (0.5-10% v/v). While FBS is the most
commonly applied supplement in animal cell culture media, other
serum sources are also routinely used, including newborn calf,
horse and human. Organs or glands that have been used to prepare
extracts for the supplementation of culture media include
submaxillary gland (Cohen, S., J. Biol. Chem. 237:1555-1565
(1961)), pituitary (Peehl, D. M., and Ham, R. G., In Vitro
16:516-525 (1980); U.S. Pat. No. 4,673,649), hypothalamus (Maciag,
T., et al., Proc. Natl. Acad. Sci. USA 76:5674-5678 (1979);
Gilchrest, B. A., et al., J. Cell. Physiol. 120:377-383 (1984)),
ocular retina (Barretault, D., et al., Differentiation 18:29-42
(1981)) and brain Maciag, T., et al., Science 211:1452-1454
(1981)). These types of chemically undefined supplements serve
several useful functions in cell culture media (Lambert, K. J. et
al., In: Animal Cell Biotechnology, Vol. 1, Spier, R. E. et al.,
Eds., Academic Press New York, pp. 85-122 (1985)). For example,
these supplements provide carriers or chelators for labile or
water-insoluble nutrients; bind and neutralize toxic moieties;
provide hormones and growth factors, protease inhibitors and
essential, often unidentified or undefined low molecular weight
nutrients; and protect cells from physical stress and damage. Thus,
serum or organ/gland extracts are commonly used as relatively
low-cost supplements to provide an optimal culture medium for the
cultivation of animal cells.
Methods of Production of Culture Media
[0016] Culture media are typically produced in liquid form or in
powdered form. Each of these forms has particular advantages and
disadvantages.
[0017] For example, liquid culture medium has the advantage that it
is provided ready-to-use (unless supplementation with nutrients or
other components is necessary or desired), and that the
formulations have frequently been optimized for particular cell
types. Liquid media have the disadvantages, however, that they
often do require the addition of supplements (e.g., L-glutamine,
serum, extracts, cytokines, lipids, etc.) for optimal performance
in cell cultivation. Furthermore, liquid medium is often difficult
to sterilize economically, since many of the components are heat
labile (thus obviating the use of autoclaving, for example) and
bulk liquids are not particularly amenable to penetrating
sterilization methods such as gamma or ultraviolet irradiation;
thus, liquid culture media are most often sterilized by filtration,
which can become a time-consuming and expensive process.
Furthermore, production and storage of large batch sizes (e.g.,
1000 liters or more) of liquid culture media are impractical, and
the components of liquid culture media often have relatively short
shelf lives.
[0018] To overcome some of these disadvantages, liquid culture
medium can be formulated in concentrated form; these media
components may then be diluted to working concentrations prior to
use. This approach provides the capability of making larger and
variable batch sizes than with standard culture media, and the
concentrated media formulations or components thereof often have
longer shelf-life (see U.S. Pat. No. 5,474,931, which is directed
to culture media concentrate technology). Despite these advantages,
however, concentrated liquid media still have the disadvantages of
their need for the addition of supplements (e.g., FBS, L-glutamine
or organ/gland extracts), and may be difficult to sterilize
economically.
[0019] As an alternative to liquid media, powdered culture media
are often used. The powders are reconstituted by dilution in
solvent to produce a reconstituted liquid, e.g., a liquid medium or
medium supplement.
[0020] Powdered media are typically produced by admixing the dried
components of the culture medium via a mixing process, e.g.,
ball-milling (also referred to herein, and interchangeably, as
"Fitzmilling"), or by lyophilizing pre-made liquid culture medium.
This approach has the advantages that even larger batch sizes may
be produced, the powdered media typically have longer shelf lives
than liquid media, and the media can be sterilized by irradiation
(e.g., gamma or ultraviolet irradiation) or ethylene oxide
permeation after formulation. However, powdered media have several
distinct disadvantages. For example, some of the components of
powdered media become insoluble or aggregate upon lyophilization
such that resolubilization is difficult or impossible. Furthermore,
powdered media typically comprise fine dust particles which can
make them particularly difficult to reconstitute without some loss
of material, and which may further make them impractical for use in
many biotechnology production facilities operating under GMP/GLP,
USP or ISO 9000 settings. Additionally, many of the supplements
used in culture media, e.g., L-glutamine and FBS, cannot be added
to the culture medium prior to lyophilization or ball-milling due
to their instability or propensity to aggregate upon concentration
or due to their sensitivity to shearing by processes such as
ball-milling. Furthermore, many of these supplements, particularly
serum supplements such as FBS, show a substantial loss of activity
or are rendered completely inactive if attempts are made to produce
powdered supplements by processes such as lyophilization. Finally,
powdered media typically do not contain bicarbonate buffering
systems and require post-reconstitution adjustment of pH, while
components required in .quadrature.g/ml amounts, or less, are
typically added post-reconstitution because of homogeneity
concerns.
[0021] Thus, there exists a current need for rapidly dissolving
nutritionally complex stable dry powder nutritive media, media
supplements, media subgroups and buffers, which can be prepared in
variable bulk quantities and which are amenable to sterilization
particularly by ionizing or ultraviolet irradiation. By use of the
present invention, cell culture media can be manufactured such that
no additional manipulations are needed other than adding solvent
and solubilizing the media components.
BRIEF SUMMARY OF THE INVENTION
[0022] The present invention provides methods for the production of
nutritive media, media supplement, media subgroup and buffer
powders comprising agglomerating a dry powder nutritive media,
media supplement, media subgroup or buffer with a solvent. The
invention also relates to methods for the production of powdered
nutritive media, media supplements, media subgroups, and buffers,
comprising spray-drying a liquid nutritive medium, medium
supplement, medium subgroup or buffer under conditions sufficient
to produce their dry powder counterparts. Such conditions may, for
example, comprise controlling heat and humidity until the powdered
media, media supplement, media subgroup or buffer is formed.
According to the invention, the method may further comprise
sterilizing the nutritive media, media supplement, media subgroup
or buffer powder, which may be accomplished prior to or after
packaging the powder. In particularly preferred methods, the
sterilization is accomplished after packaging of the powder by
irradiation of the packaged powder with gamma rays.
[0023] Particularly preferred nutritive medium powders that may be
produced according to the invention include culture medium powders
selected from the group consisting of a bacterial culture medium
powder, a yeast culture medium powder, a plant culture medium
powder and an animal culture medium powder.
[0024] Particularly preferred media supplements that may be
produced by the methods of the invention include: powdered animal
sera, such as bovine sera (e.g., fetal bovine, newborn calf or
normal calf sera), human sera, equine sera, porcine sera, monkey
sera, ape sera, rat sera, murine sera, rabbit sera, ovine sera and
the like; cytokines (including growth factors (such as EGF, aFGF,
bFGF, KGF, HGF, IGF-1, IGF-2, NGF and the like), interleukins,
colony-stimulating factors and interferons); attachment factors or
extracellular matrix components (such as collagens, laminins,
proteoglycans, glycosaminoglycans, fibronectin, vitronectin and the
like); lipids (such as phospholipids, cholesterol, bovine
cholesterol concentrate, fatty acids, sphingolipids and the like);
and extracts of animal tissues, organs or glands (such as bovine
pituitary extract, bovine brain extract, chick embryo extract,
bovine embryo extract, chicken meat extract, achilles tendon and
extracts thereof) and the like). Other media supplements that may
be produced by the present methods include a variety of proteins
(such as serum albumins, particularly bovine or human serum
albumins; immunoglobulins and fragments or complexes thereof;
aprotinin; hemoglobin; haemin or haematin; enzymes (such as
trypsin, collagenases, pancreatinin or dispase); lipoproteins;
ferritin; etc.) which may be natural or recombinant; vitamins
(including but not limited to vitamins A, B.sub.1, B.sub.2,
B.sub.3, B.sub.6 B.sub.12, C, D, E, K and H (biotin)); amino acids
and variants thereof (including, but not limited to, L-glutamine
and cystine), enzyme co-factors, trace elements (such as calcium,
copper, iron, magnesium, manganese, nickel, potassium, tin, zinc,
selenium, vanadium and the like), and other components useful in
cultivating cells in vitro that will be familiar to one of ordinary
skill.
[0025] The invention also provides complete dry powder culture
media formulations that support the cultivation of cells in vitro
upon reconstitution of the medium with a solvent, without the need
for the addition of any supplemental nutrient components to the
medium prior to use. In accordance with the invention, such
complete media may be automatically pH-adjusting media, and may
comprise one or more components such as serum, one or more culture
medium supplements, L-glutamine, insulin, transferrin, one or more
hormones, one or more lipids, one or more growth factors, one or
more cytokines, one or more neurotransmitters, one or more extracts
of animal tissues, organs or glands, one or more enzymes, one or
more proteins, one or more trace elements, one or more
extracellular matrix components, one or more antibiotics, one or
more viral inhibitors, and or one or more buffers.
[0026] The nutritive media and media supplements prepared by the
invention may also comprise subgroups such as serum (preferably
those described above), L-glutamine, insulin, transferrin, one or
more lipids (preferably one or more phospholipids, sphingolipids,
fatty acids or cholesterol), one or more cytokines (preferably
those described above), one or more neurotransmitters, one or more
extracts of animal tissues, organs or glands (preferably those
described above), one or more proteins (preferably those described
above) or one or more buffers (preferably sodium bicarbonate), or
any combination thereof.
[0027] Buffer powders particularly suitable for preparation
according to the methods of the invention include buffered saline
powders, most particularly phosphate-buffered saline powders or
Tris-buffered saline powders. In particular, the invention provides
methods of preparing "auto-pH" buffer powders which automatically
are at a desired pH upon rehydration with a solvent.
[0028] The invention also provides nutritive medium powders, medium
supplement powders (including powders of the above-described
supplements) and buffer powders, particularly auto-pH medium,
medium supplement and buffer powders, prepared according to these
methods.
[0029] The invention also relates to methods of preparing dried
cells, including prokaryotic (e.g., bacterial) and eukaryotic
(e.g., fungal (especially yeast), animal (especially mammalian,
including human) and plant) cells, comprising obtaining a cell to
be dried, contacting the cell with one or more stabilizers (e.g., a
polysaccharide such as trehalose), forming an aqueous suspension
comprising the cell, and spray-drying the cell suspension under
conditions favoring the production of a dried powder. The invention
also relates to dried cell powders produced by these methods.
[0030] The invention further relates to methods of preparing
sterile powdered culture media, media supplements, media subgroups
and buffers. One such method comprises exposing the above-described
powdered culture media, media supplements, media subgroups and
buffers to y irradiation such that bacteria, fungi, spores and
viruses that may be resident in the powders are rendered incapable
of replication. In a preferred such method, the powdered media,
media supplements, media subgroups and buffers are y irradiated at
a total dosage of about 10-100 kilograys (kGy), preferably a total
dosage of about 15-75 kGy, 15-50 kGy, 15-40 kGy or 20-40 kGy, more
preferably a total dosage of about 20-30 kGy, and most preferably a
total dosage of about 25 kGy, for about 1 hour to about 7 days,
preferably for about 1 hour to about 5 days, more preferably for
about 1 hour to about 3 days, about 1 hour to about 24 hours or
about 1-5 hours, and most preferably about 1-3 hours. The invention
also relates to sterile powdered culture media, media supplements,
media subgroups and buffers produced by these methods.
[0031] The invention further provides methods of culturing a cell
comprising reconstituting the nutritive media, media supplement,
media subgroup or buffer of the invention with a solvent, which
preferably comprises serum or water, and contacting the cell with
the reconstituted nutritive media, media supplement, media subgroup
or buffer under conditions favoring the cultivation of the cell.
Any cell may be cultured according to the present methods,
particularly bacterial cells, yeast cells, plant cells or animal
cells. Preferable animal cells for culturing by the present methods
include insect cells (most preferably Drosophila cells, Spodoptera
cells and Trichoplusa cells), nematode cells (most preferably C.
elegans cells) and mammalian cells (most preferably CHO cells, COS
cells, VERO cells, BHK cells, AE-1 cells, SP2/0 cells, L5.1 cells,
hybridoma cells or human cells). Cells cultured according to this
aspect of the invention may be normal cells, diseased cells,
transformed cells, mutant cells, somatic cells, germ cells, stem
cells, precursor cells or embryonic cells, any of which may be
established or transformed cell lines or obtained from natural
sources.
[0032] The invention also provides compositions comprising one or
more of the culture media, media supplement, media subgroup or
buffer powders of the invention and at least one cell. Such
compositions may comprise, for example, an automatically
pH-adjusting culture medium powder of the invention or a complete
dry powder medium of the invention and one or more cells, such as
one or more bacterial cells, one or more plant cells, one or more
yeast cells, and one or more animal cells (including but not
limited to one or more mammalian cells such as one or more human
cells). Compositions according to this aspect of the invention may
be in powder form which, upon reconstitution with a solvent,
produce an active culture of the one or more cells contained in the
composition.
[0033] The invention is further directed to kits for use in the
cultivation of a cell. Kits according to the invention may comprise
one or more containers containing one or more of the nutritive
media powders, media supplement powders, media subgroup powders or
buffer powders of the invention, or any combination thereof. The
kits may also comprise one or more cells or cell types, including
the dried cell powders of the invention.
[0034] The invention specifically provides the following
aspects:
[0035] Aspect 1. A method for producing an automatically
pH-adjusting dry powdered culture medium, comprising: (a)
determining the ratio of pH-opposing forms of buffer salts required
to be added to said powder to automatically provide a desired final
pH upon reconstitution of said powder with a solvent; and (b)
adding amounts of pH-opposing forms of buffer salts to said powder
in the ratio determined in step (a).
[0036] Aspect 2. The method of aspect 1, further comprising
packaging said dry powdered medium.
[0037] Aspect 3. The method of aspect 1, further comprising
sterilizing said dry powdered medium.
[0038] Aspect 4. The method of aspect 3, wherein said sterilization
accomplished by irradiating said dry powdered medium with gamma
rays until said medium is sterile.
[0039] Aspect 5. The method of any one of aspects 1-3, wherein said
medium comprises at least one monobasic and/or dibasic buffering
salt.
[0040] Aspect 6. The method of aspect 5, wherein said monobasic
and/or dibasic buffering salt is a monobasic and/or dibasic
phosphate salt.
[0041] Aspect 7. The method of aspect 6, wherein at least one of
said monobasic and/or dibasic phosphate salts is a sodium phosphate
salt.
[0042] Aspect 8. The method of aspect 6, wherein at least one of
said monobasic or dibasic phosphate salts is a potassium phosphate
salt.
[0043] Aspect 9. The method of aspect 1, wherein said dry powder
medium contains sodium bicarbonate but does not liberate CO.sub.2
upon storage.
[0044] Aspect 10. An automatically pH-adjusting dry powdered
culture medium produced by the method of any one of aspects 1-3 and
9.
[0045] Aspect 11. A complete dry powder culture medium that
supports the cultivation of a cell in vitro upon reconstitution of
the medium with a solvent without the addition of any supplemental
nutrient components to said medium.
[0046] Aspect 12. The medium of aspect 11, wherein said medium is
an automatically pH-adjusting medium.
[0047] Aspect 13. The medium of aspect 11, wherein said medium
comprises one or more components selected from the group of
components consisting of serum, one or more culture medium
supplements, L-glutamine, insulin, transferrin, one or more
hormones, one or more lipids, one or more growth factors, one or
more cytokines, one or more neurotransmitters, one or more extracts
of animal tissues, organs or glands, one or more enzymes, one or
more proteins, one or more trace elements, one or more
extracellular matrix components, one or more antibiotics, one or
more viral inhibitors, and or one or more buffers.
[0048] Aspect 14. A method of cultivating a cell, comprising
reconstituting an automatically pH-adjusting dry powdered medium
with a solvent to form a culture medium solution, and contacting
the cell with said liquid solution under conditions favoring the
cultivation of the cell.
[0049] Aspect 15. A method of cultivating a cell comprising
preparing an automatically pH-adjusting dry powdered culture medium
prepared according to the method any one of aspects 1-3 and 9,
reconstituting the medium with at least one solvent to form a
culture medium solution, and contacting a cell with said solution
under conditions favoring cultivation of the cell.
[0050] Aspect 16. A method of cultivating a cell, comprising
reconstituting the culture medium of aspect 10 with a solvent to
form a culture medium solution, and contacting the cell with said
solution under conditions favoring the cultivation of the cell.
[0051] Aspect 17. A method of cultivating a cell, comprising
reconstituting the culture medium of aspect 11 with a solvent to
form a culture medium solution, and contacting the cell with said
solution under conditions favoring the cultivation of the cell.
[0052] Aspect 18. The method of any one of aspects 14, 16 and 17,
wherein said cell is a bacterial cell.
[0053] Aspect 19. The method of aspect 15, wherein said cell is a
bacterial cell.
[0054] Aspect 20. The method of any one of aspects 14, 16 and 17,
wherein said cell is a eukaryotic cell.
[0055] Aspect 21. The method of aspect 15, wherein said cell is a
eukaryotic cell.
[0056] Aspect 22. The method of aspect 20, wherein said eukaryotic
cell is a yeast cell, a plant cell, or a cell line derived
therefrom.
[0057] Aspect 23. The method of aspect 21, wherein said eukaryotic
cell is a yeast cell, a plant cell, or a cell line derived
therefrom.
[0058] Aspect 24. The method of aspect 20, wherein said eukaryotic
cell is an animal cell or a cell line derived therefrom.
[0059] Aspect 25. The method of aspect 21, wherein said eukaryotic
cell is an animal cell or a cell line derived therefrom.
[0060] Aspect 26. The method of aspect 24 or aspect 25, wherein
said animal cell is a mammalian cell or a cell line derived
therefrom.
[0061] Aspect 27. The method of aspect 26, wherein said mammalian
cell is a human cell or a cell line derived therefrom.
[0062] Aspect 28. A kit for culturing a cell, comprising one or
more containers containing an automatically pH-adjusting dry
powdered culture medium prepared according to the method of any one
of aspects 1-3 and 9.
[0063] Aspect 29. A kit for culturing a cell, comprising one or
more containers containing the automatically pH-adjusting dry
powdered culture medium of aspect 10.
[0064] Aspect 30. A kit for culturing a cell, comprising one or
more containers containing the complete dry powdered culture medium
of aspect 11.
[0065] Aspect 31. The kit of aspect 28, wherein said kit further
comprises one or more additional containers containing at least one
additional component selected from the group consisting of at least
one growth factor, at least one culture medium supplement, at least
one animal tissue extract, at least one animal organ extract, at
least one animal gland extract, at least one enzyme, at least one
protein, at least one vitamin, at least one cytokine, at least one
lipid, at least one trace element, at least one extracellular
matrix component, at least one buffer, at least one antibiotic, and
at least one viral inhibitor.
[0066] Aspect 32. The kit of aspect 29 or aspect 30, wherein said
kit further comprises one or more additional containers containing
at least one additional component selected from the group
consisting of at least one growth factor, at least one culture
medium supplement, at least one animal tissue extract, at least one
animal organ extract, at least one animal gland extract, at least
one enzyme, at least one protein, at least one vitamin, at least
one cytokine, at least one lipid, at least one trace element, at
least one extracellular matrix component, at least one buffer, at
least one antibiotic, and at least one viral inhibitor.
[0067] Aspect 33. A composition comprising the automatically
pH-adjusting culture medium of aspect 10 and at least one cell.
[0068] Aspect 34. The composition of aspect 33, wherein said
composition is a powder.
[0069] Aspect 35. A composition comprising the complete culture
medium of aspect 11 and at least one cell.
[0070] Aspect 36. The composition of aspect 33 or aspect 35,
wherein said cell is selected from the group consisting of a
bacterial cell, a yeast cell, a plant cell and an animal cell.
[0071] Aspect 37. The composition of aspect 36, wherein said animal
cell is a mammalian cell.
[0072] Aspect 38. The composition of aspect 37, wherein said
mammalian cell is a human cell.
[0073] Aspect 39. The composition of aspect 36, wherein said cell
is an established or transformed cell line.
[0074] Other preferred embodiments of the present invention will be
apparent to one of ordinary skill in light of the following
drawings and description of the invention, and of the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] FIG. 1 is a histogram of a densitometric scan of SDS-PAGE of
samples of fetal bovine serum (FBS) prepared in powdered form by
the methods of the invention (FIG. 1A) and conventional liquid FBS
(FIG. 1B).
[0076] FIG. 2 is a composite of line graphs of growth (FIG. 2A) and
passage success (FIG. 2B) of SP2/0 cells in Dulbecco's Modified
Eagle's Medium (DMEM) supplemented with 2% (w/v) FBS prepared in
powdered form by the agglomeration methods of the invention.
[0077] FIG. 3 is composite of histograms of spectrophotometric
scans (.lamda.=200-350 nm) of powdered fetal bovine serum (FBS)
prepared by spray-drying according to the methods of the invention
(FIG. 3A) or of standard liquid FBS (FIG. 3B).
[0078] FIG. 4 is a composite of line graphs showing the pH
titration (buffer capacity), on two different dates (FIGS. 4A and
4B), of various dry powdered media (DPM) prepared by the methods of
the invention or by ball-milling, with or without the addition of
sodium bicarbonate.
[0079] FIG. 5 is a composite of bar graphs showing the effect of
agglomeration on dissolution rates (in water) of Opti-MEM I.TM.
(FIG. 5A) or DMEM (FIG. 5B). Media were agglomerated with water or
FBS as indicated.
[0080] FIG. 6 is a composite of line graphs showing growth over
seven days of SP2/0 cells in agglomerated Opti-MEM I.TM. (FIG. 6A)
or DMEM (FIG. 6B), both containing 2% FBS.
[0081] FIG. 7 is a composite of line graphs showing growth over
seven days of SP2/0 cells (FIG. 7A), AE-1 cells (FIG. 7B) and L5.1
cells (FIG. 7C) in agglomerated DMEM containing 10% FBS.
[0082] FIG. 8 is a composite of line graphs showing passage success
of SP2/0 cells in Opti-MEM I.TM. (FIG. 8A) or DMEM (FIG. 8B),
agglomerated with either water or FBS, supplemented with 2%
FBS.
[0083] FIG. 9 is a composite of line graphs showing passage success
of SP2/0 cells (FIG. 9A), AE-1 cells (FIG. 9B) and L5.1 cells (FIG.
9C) in DMEM agglomerated with FBS and sodium bicarbonate and
supplemented with 10% FBS.
[0084] FIG. 10 is a line graph showing the growth of SP2/0 cells
over four passages in standard water-reconstituted powdered culture
media (control media), or in agglomerated powdered culture media
prepared in large-scale amounts according to the methods of the
invention. Results are shown for control media (.quadrature.),
water-agglomerated powdered culture media of the invention
(.diamond-solid.) and water-agglomerated auto-pH powdered culture
media (containing sodium bicarbonate) of the invention
(.box-solid.).
[0085] FIG. 11 is a line graph of AE-1 cells cultured over six or
seven days in medium containing 2% (.DELTA.) or 10%
(.diamond-solid.) liquid fetal bovine serum (FBS), or 2% (_) or 10%
(.box-solid.) powdered FBS prepared by the spray-drying methods of
the invention. Duplicate experiments are shown in FIGS. 11A and
11B.
[0086] FIG. 12 is a line graph of SP2/0 cells cultured over seven
days in medium containing 2% (.DELTA.) or 10% (.diamond-solid.)
liquid FBS, or 2% (_) or 10% (.box-solid.) powdered FBS prepared by
the spray-drying methods of the invention. Duplicate experiments
are shown in FIGS. 12A and 12B.
[0087] FIG. 13 is a line graph of AE-1 cell growth over four
passages in media containing 5% liquid FBS (.diamond-solid.) or 5%
powdered FBS prepared by the spray-drying methods of the invention
(.box-solid.).
[0088] FIG. 14 is a line graph indicating the effect of .gamma.
irradiation and agglomeration on the growth of SP2/0 cells over
five days.
[0089] FIG. 15 is a bar graph indicating the effect of .gamma.
irradiation on the growth of VERO cells in agglomerated culture
media.
[0090] FIG. 16 is a series of line graphs indicating the effect of
.gamma. irradiation on the ability of transferrin to support the
growth of 293 cells over four passages. In each graph, cells were
cultured in standard serum-free 293 medium (.diamond-solid.), in
medium without transferrin (.box-solid.), in medium containing
powdered transferrin that had been .gamma. irradiated at
-70.degree. C. (.DELTA.) or room temperature (*), or in medium
containing powdered transferrin that had not been .gamma.
irradiated but that had been stored at -70.degree. C. (_) or at
room temperature (.circle-solid.). Results for each data point are
the averages of duplicate flasks.
[0091] FIG. 16A: passage 1 cells;
[0092] FIG. 16B: passage 2 cells;
[0093] FIG. 16C: passage 3 cells;
[0094] FIG. 16D: passage 4 cells.
[0095] FIG. 17 is a series of bar graphs indicating the effect of
.gamma. irradiation, under different irradiation conditions, on the
ability of FBS to support growth of anchorage-independent cells
(FIGS. 17A and 17B) and anchorage-dependent cells (FIGS. 17C and
17D) at first (Px1), second (Px2) and third (Px3) passages.
[0096] FIG. 17A: SP2/0 cells;
[0097] FIG. 17B: AE-1 cells;
[0098] FIG. 17C: VERO cells;
[0099] FIG. 17D: BHK cells.
[0100] FIG. 18 is a line graph depicting the buffering kinetics of
solutions of 5.1 mM sodium phosphate in the dibasic (x-x) or
monobasic (O-O) forms upon challenge with various volumes of 5N
HCl.
[0101] FIG. 19 is a series of line graphs depicting the buffering
kinetics for RPMI-1640 culture media in various forms, with or
without the addition of NaHCO.sub.3.
[0102] FIG. 19A: liquid vs. powder media.
[0103] .diamond-solid.-.diamond-solid.: liquid RPMI-1640 containing
NaHCO.sub.3 (note that this line is superimposed with that for
powder RPMI-1640 containing NaHCO.sub.3);
[0104] .box-solid.-.box-solid.: liquid RPMI-1640 with no
NaHCO.sub.3;
[0105] .DELTA.-.DELTA.: powder RPMI-1640 containing NaHCO.sub.3
(note that this line is superimposed with that for liquid RPMI-1640
containing NaHCO.sub.3);
[0106] x-x: powder RPMI-1640 containing NaHCO.sub.3, agglomerated
but without auto-pH;
[0107] *-*: powder RPMI-1640 containing NaHCO.sub.3, agglomerated
and with auto-pH.
[0108] FIG. 19B: powder media, milled or non-milled.
[0109] .diamond-solid.-.diamond-solid.: milled RPMI-1640 containing
non-milled NaHCO.sub.3 (note that this line is superimposed with
that for non-milled RPMI-1640 containing milled NaHCO.sub.3);
[0110] .box-solid.-.box-solid.: milled RPMI-1640 with no
NaHCO.sub.3;
[0111] .DELTA.-.DELTA.: non-milled RPMI-1640 containing milled
NaHCO.sub.3 (note that this line is superimposed with that for
milled RPMI-1640 containing non-milled NaHCO.sub.3);
[0112] x-x: non-milled RPMI-1640 containing milled NaHCO.sub.3,
agglomerated but without auto-pH;
[0113] *-*: non-milled RPMI-1640 containing milled NaHCO.sub.3,
agglomerated and with auto-pH.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0114] In the description that follows, a number of terms
conventionally used in the field of cell culture media are utilized
extensively. In order to provide a clear and consistent
understanding of the specification and claims, and the scope to be
given such terms, the following definitions are provided.
[0115] The term "powder" as used herein refers to a composition
that is present in granular form, which may or may not be complexed
or agglomerated with a solvent such as water or serum. The term
"dry powder" may be used interchangeably with the term "powder;"
however, "dry powder" as used herein simply refers to the gross
appearance of the granulated material and is not intended to mean
that the material is completely free of complexed or agglomerated
solvent unless otherwise indicated.
[0116] The term "ingredient" refers to any compound, whether of
chemical or biological origin, that can be used in cell culture
media to maintain or promote the growth of proliferation of cells.
The terms "component," "nutrient" and ingredient" can be used
interchangeably and are all meant to refer to such compounds.
Typical ingredients that are used in cell culture media include
amino acids, salts, metals, sugars, lipids, nucleic acids,
hormones, vitamins, fatty acids, proteins and the like. Other
ingredients that promote or maintain cultivation of cells ex vivo
can be selected by those of skill in the art, in accordance with
the particular need.
[0117] Mean diameter. Particle size quantified by laser light
scattering or by mechanical segregation on relative mesh size
calibrated screens.
[0118] Homogenous Mixture. As used herein homogenous mixture is a
mixture whose composition when samples from various location varies
less than 5.0% the relative standard deviation (RDS) of the actual
concentration from the theoretical concentration. Each sample may
be as large as desired, for example, up to 1% 10% or 20% of the
total, but can be quite small, for example an 20 gm sample of 140
kg lot (0.015%) or 20 gm sample of 250 kg lot (0.008%) or as small
a samples as 20 gm sample of 500 kg lot (0.0004%). Analytical
limits and particle size will put practical limits the minimum
desired sample size.
[0119] Physiologic pH. As used herein physiologic pH is greater
than about 4 and less than about 9. Other pH values or ranges,
e.g., minimum or maximum pHs of greater than 4.2, 4.5, 4.8, 5.0,
5.2, 5.5, 5,7, 5.8, 6.0, 6.2, 6.5, 6,7, 6.8, 7.0, 7.2, 7.4, 7.5,
7.8, 8.0, 8.2, 8.4, 8.5, 8.7, 8.8, etc. may also be used for
dissolving supplements. Some supplements, though not preferred, may
only be entirely soluble outside these ranges.
[0120] Polar solvent. As used herein polar solvent may include
water, saline, water with soluble acid or base ions, with a pH
range of 1.0-10.0, stabilizers, surfactants, preservatives, and
alcohols and other non polar organic solvents.
[0121] The term "cytokine" refers to a compound that induces a
physiological response in a cell, such as growth, differentiation,
senescence, apoptosis, cytotoxicity or antibody secretion. Included
in this definition of "cytokine" are growth factors, interleukins,
colony-stimulating factors, interferons and lymphokines.
[0122] By "cell culture" or "culture" is meant the maintenance of
cells in an artificial, e.g., an in vitro environment. It is to be
understood, however, that the term "cell culture" is a generic term
and may be used to encompass the cultivation not only of individual
prokaryotic (e.g., bacterial) or eukaryotic (e.g., animal, plant
and fungal) cells, but also of tissues, organs, organ systems or
whole organisms, for which the terms "tissue culture," "organ
culture," "organ system culture" or "organotypic culture" may
occasionally be used interchangeably with the term "cell
culture."
[0123] By "cultivation" is meant the maintenance of cells in an
artificial environment under conditions favoring growth,
differentiation or continued viability, in an active or quiescent
state, of the cells. Thus, "cultivation" may be used
interchangeably with "cell culture" or any of its synonyms
described above.
[0124] By "culture vessel" is meant a glass, plastic, or metal
container that can provide an aseptic environment for culturing
cells.
[0125] The phrases "cell culture medium," "culture medium" (plural
"media" in each case) and "medium formulation" refer to a nutritive
solution that supports the cultivation and/or growth of cells;
these phrases maybe used interchangeably.
[0126] By "extract" is meant a composition comprising a
concentrated preparation of the subgroups of a substance, typically
formed by treatment of the substance either mechanically (e.g., by
pressure treatment) or chemically (e.g., by distillation,
precipitation, enzymatic action or high salt treatment).
[0127] By "enzymatic digest" is meant a composition comprising a
specialized type of extract, namely one prepared by treating the
substance to be extracted (e.g., plant components or yeast cells)
with at least one enzyme capable of breaking down the components of
the substance into simpler forms (e.g., into a preparation
comprising mono- or disaccharides and/or mono-, di- or
tripeptides). In this context, and for the purposes of the present
invention, the term "hydrolysate" may be used interchangeably with
the term "enzymatic digest."
[0128] The term "contacting" refers to the placing of cells to be
cultivated into a culture vessel with the medium in which the cells
are to be cultivated. The term "contacting" encompasses mixing
cells with medium, pipetting medium onto cells in a culture vessel,
and submerging cells in culture medium.
[0129] The term "combining" refers to the mixing or admixing of
ingredients in a cell culture medium formulation.
[0130] The term "pillowing" refers to the event which occurs when
any moisture, including atmospheric water, infiltrates a container
and moistens the powder contained therein. Such moistening may
result in acidic conditions within the container that will cause
the liberation of CO.sub.2 gas from the powder ("off-gassing").
When dry powder "pillows" in a sealed container, the off-gassing
may cause the container to swell to the point of bursting.
[0131] The term "small-quantity" refers to components present in
the medium in .mu.g/ml, .mu.g/L, or lower amounts.
[0132] A cell culture medium is composed of a number of ingredients
and these ingredients vary from one culture medium to another. A
"1.times. formulation" is meant to refer to any aqueous solution
that contains some or all ingredients found in a cell culture
medium at working concentrations. The "1.times. formulation" can
refer to, for example, the cell culture medium or to any subgroup
of ingredients for that medium. The concentration of an ingredient
in a 1.times. solution is about the same as the concentration of
that ingredient found in a cell culture formulation used for
maintaining or cultivating cells in vitro. A cell culture medium
used for the in vitro cultivation of cells is a 1.times.
formulation by definition. When a number of ingredients are
present, each ingredient in a 1.times. formulation has a
concentration about equal to the concentration of those ingredients
in a cell culture medium. For example, RPMI-1640 culture medium
contains, among other ingredients, 0.2 g/L L-arginine, 0.05 g/L
L-asparagine, and 0.02 g/L L-aspartic acid. A "1.times.
formulation" of these amino acids contains about the same
concentrations of these ingredients in solution. Thus, when
referring to a "1.times. formulation," it is intended that each
ingredient in solution has the same or about the same concentration
as that found in the cell culture medium being described. The
concentrations of ingredients in a 1.times. formulation of cell
culture medium are well known to those of ordinary skill in the
art. See Methods For Preparation of Media, Supplements and
Substrate For Serum-Free Animal Cell Culture Allen R. Liss, N.Y.
(1984), which is incorporated by reference herein in its entirety.
The osmolality and/or pH, however, may differ in a 1.times.
formulation compared to the culture medium, particularly when fewer
ingredients are contained in the 1.times. formulation.
[0133] A "supplement" is a cell nutrient, cell modifier, cell
growth modulator, etc. that is added to a cell culture medium. A
supplement may have no ingredients in common with the medium being
supplemented or may have one or more ingredients in common. The
supplement will differ from the medium being supplemented in at
least one manner, such as a different concentration of one or more
ingredients, for example a different ratio of two ingredients, a
different ingredient mix, additional ingredients or omitted
ingredients in the supplement. For example a supplement may omit
salts to the extent feasible and may contain, for example,
significantly enhanced concentrations of growth factors or amino
acids. A preferred supplement formulation contains at least 2, more
preferably 3, but perhaps at least 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, or more amino acids including salts or dimers thereof
.
[0134] A "10.times. formulation" is meant to refer to a solution
wherein each ingredient in that solution is about 10 times more
concentrated than the same ingredient in the cell culture medium.
For example, a 10.times. formulation of RPMI-1640 culture medium
may contain, among other ingredients, 2.0 g/L L-arginine, 0.5 g/L
L-asparagine, and 0.2 g/L L-aspartic acid (compare 1.times.
formulation, above). A "10.times. formulation" may contain a number
of additional ingredients at a concentration about 10 times that
found in the 1.times. culture medium. As will be readily apparent,
"20.times. formulation," "25.times. formulation," "50.times.
formulation" and "100.times. formulation" designate solutions that
contain ingredients at about 20-, 25-, 50- or 100- fold
concentrations, respectively, as compared to a 1.times. cell
culture medium. Again, the osmolality and pH of the media
formulation and concentrated solution may vary. See U.S. Pat. No.
5,474,931, which is directed to culture media concentrate
technology.
[0135] An "auto-pH" powder of the invention (e.g., auto-pH medium,
medium supplement or buffer powder) is a powder which has been
formulated such that, upon rehydration with a solvent, the
resulting medium, medium supplement or buffer solution is at a
desired pH and does not require adjustment of the pH with acid or
base prior to use. For example, an auto-pH culture medium that is
formulated to be used at pH 7.4 will, upon rehydration with a
solvent, be at pH 7.4 and therefore will be ready for immediate use
without adjustment of pH. Such auto-pH powders of the invention may
also be referred to herein interchangeably as "automatically
pH-adjusting" powders.
Overview
[0136] The present invention is directed to methods of producing
nutritive media, media supplements, media subgroups or buffers.
Nutritive media, media supplements and media subgroups produced by
the present methods are any media, media supplement or media
subgroup (serum-free or serum-containing) which may be used to
support the growth of a cell, which may be a bacterial cell, a
fungal cell (particularly a yeast cell), a plant cell or an animal
cell (particularly an insect cell, a nematode cell or a mammalian
cell, most preferably a human cell), any of which may be a somatic
cell, a germ cell, a normal cell, a diseased cell, a transformed
cell, a mutant cell, a stem cell, a precursor cell or an embryonic
cell. Preferred such nutritive media include, but are not limited
to, cell culture media, most preferably a bacterial cell culture
medium, plant cell culture medium or animal cell culture medium.
Preferred media supplements include, but are not limited to,
undefined supplements such as extracts of bacterial, animal or
plant cells, glands, tissues or organs (particularly bovine
pituitary extract, bovine brain extract and chick embryo extract);
and biological fluids (particularly animal sera, and most
preferably bovine serum (particularly fetal bovine, newborn calf or
normal calf serum), horse serum, porcine serum, rat serum, murine
serum, rabbit serum, monkey serum, ape serum or human serum, any of
which may be fetal serum) and extracts thereof (more preferably
serum albumin and most preferably bovine serum albumin or human
serum albumin). Medium supplements may also include defined
replacements such as LipoMAX.RTM., OptiMAb.RTM., Knock-Out.TM. SR
(each available from Invitrogen Corporation, Carlsbad, Calif.), and
the like, which can be used as substitutes for the undefined media
supplements described above. Such supplements may also comprise
defined components, including but not limited to, hormones,
cytokines, neurotransmitters, lipids, attachment factors, proteins
and the like.
[0137] Nutritive media can also be divided into various subgroups
(see U.S. Pat. No. 5,474,931) which can be prepared by, and used in
accordance with, the methods of the invention. Such subgroups can
be combined to produce the nutritive media of the present
invention.
[0138] By the methods of the present invention, any nutritive
media, media supplement, media subgroup or buffer may be produced
and stored for an extended period of time without significant loss
of biological and biochemical activity. By "without significant
loss of biological and biochemical activity" is meant a decrease of
less than about 30%, preferably less than about 25%, more
preferably less than about 20%, still more preferably less than
about 15%, and most preferably less than about 10%, of the
biological or biochemical activity of the nutritive media, media
supplement, media subgroup or buffer when compared to a freshly
made nutritive media, media supplement, media subgroup or buffer of
the same formulation. By an "extended period of time" is meant a
period of time longer than that for which a nutritive medium,
supplement, subgroup or buffer is stored when prepared by
traditional methods such as ball-milling. As used herein, an
"extended period of time" therefore means about 1-36 months, about
2-30 months, about 3-24 months, about 6-24 months, about 9-18
months, or about 4-12 months, under a given storage condition,
which may include storage at temperatures of about -70.degree. C.
to about 25.degree. C., about -20.degree. C. to about 25.degree.
C., about 0.degree. C. to about 25.degree. C., about 4.degree. C.
to about 25.degree. C., about 10.degree. C. to about 25.degree. C.,
or about 20.degree. C. to about 25.degree. C. Assays for
determining the biological or biochemical activity of a nutritive
media, media supplement, media subgroup or buffer are well-known in
the art and are familiar to one of ordinary skill.
Formulation of Media, Media Supplements, Media Subgroups and
Buffers
[0139] Any nutritive media, media supplement, media subgroup or
buffer may be prepared by the methods of the present invention.
Particularly preferred nutritive media, media supplements and media
subgroups that may be prepared according to the invention include
cell culture media, media supplements and media subgroups that
support the growth of animal cells, plant cells, bacterial cells or
yeast cells. Particularly preferred buffers that may be prepared
according to the invention include balanced salt solutions which
are isotonic for animal cells, plant cells, bacterial cells or
yeast cells.
[0140] Examples of animal cell culture media that may be prepared
according to the present invention include, but are not limited to,
DMEM, RPMI-1640, MCDB 131, MCDB 153, MDEM, IMDM, MEM, M199, McCoy's
5A, Williams' Media E, Leibovitz's L-15 Medium, Grace's Insect
Medium, IPL-41 Insect Medium, TC-100 Insect Medium, Schneider's
Drosophila Medium, Wolf & Quimby's Amphibian Culture Medium,
cell-specific serum-free media (SFM) such as those designed to
support the culture of keratinocytes, endothelial cells,
hepatocytes, melanocytes, etc., F10 Nutrient Mixture and F12
Nutrient Mixture. Other media, media supplements and media
subgroups suitable for preparation by the invention are available
commercially (e.g., from Invitrogen Corporation, Carlsbad, Calif.,
and Sigma; St. Louis, Mo.). Formulations for these media, media
supplements and media subgroups, as well as many other commonly
used animal cell culture media, media supplements and media
subgroups are well-known in the art and may be found, for example
in the GIBCO/BRL Catalogue and Reference Guide (Invitrogen
Corporation, Carlsbad, Calif.) and in the Sigma Animal Cell
Catalogue (Sigma; St. Louis, Mo.).
[0141] Examples of plant cell culture media that may be prepared
according to the present invention include, but are not limited to,
Anderson's Plant Culture Media, CLC Basal Media, Gamborg's Media,
Guillard's Marine Plant Culture Media, Provasoli's Marine Media,
Kao and Michayluk's Media, Murashige and Skoog Media, McCown's
Woody Plant Media, Knudson Orchid Media, Lindemann Orchid Media,
and Vacin and Went Media. Formulations for these media, which are
commercially available, as well as for many other commonly used
plant cell culture media, are well-known in the art and may be
found for example in the Sigma Plant Cell Culture Catalogue (Sigma;
St. Louis, Mo.).
[0142] Examples of bacterial cell culture media that may be
prepared according to the present invention include, but are not
limited to, Trypticase Soy Media, Brain Heart Infusion Media, Yeast
Extract Media, Peptone-Yeast Extract Media, Beef Infusion Media,
Thioglycollate Media, Indole-Nitrate Media, MR-VP Media, Simmons'
Citrate Media, CTA Media, Bile Esculin Media, Bordet-Gengou Media,
Charcoal Yeast Extract (CYE) Media, Mannitol-salt Media,
MacConkey's Media, Eosin-methylene blue (EMB) media, Thayer-Martin
Media, Salmonella-Shigella Media, and Urease Media. Formulations
for these media, which are commercially available, as well as for
many other commonly used bacterial cell culture media, are
well-known in the art and may be found for example in the DIFCO
Manual (DIFCO; Norwood, Mass.) and in the Manual of Clinical
Microbiology (American Society for Microbiology, Washington,
D.C.).
[0143] Examples of fungal cell culture media, particularly yeast
cell culture media, that may be prepared according to the present
invention include, but are not limited to, Sabouraud Media and
Yeast Morphology Media (YMA). Formulations for these media, which
are commercially available, as well as for many other commonly used
yeast cell culture media, are well-known in the art and may be
found for example in the DIFCO Manual (DIFCO; Norwood, Mass.) and
in the Manual of Clinical Microbiology (American Society for
Microbiology, Washington, D.C.).
[0144] As the skilled artisan will appreciate, any of the above
media of the invention may also include one or more additional
components, such as indicating or selection agents (e.g., dyes,
antibiotics, amino acids, enzymes, substrates and the like),
filters (e.g., charcoal), salts, polysaccharides, ions, detergents,
stabilizers, and the like.
[0145] In a particularly preferred embodiment of the invention, the
above-described culture media may comprise one or more buffer
salts, preferably sodium bicarbonate, at concentrations sufficient
to provide optimal buffering capacity for the culture medium.
According to one aspect of the invention, a buffer salt, such as
sodium bicarbonate, may be added in powdered form to the powdered
medium prior to, during or following agglomeration of the medium.
In one example of this aspect of the invention, the sodium
bicarbonate maybe added to the culture medium prior to, during or
following agglomeration with an appropriate solvent (such as water,
serum or a pH-adjusting agent such as an acid (e.g., HCl at a
concentration of 1M to 5M, preferably at 1M) or a base (e.g., NaOH
at a concentration of 1M to 5M, preferably at 1M) such that, upon
reconstitution of the agglomerated medium the culture medium is at
the optimal or substantially optimal pH for cultivation of a
variety of cell types. For example, bacterial cell culture media
prepared by the present methods will, upon reconstitution,
preferably have a pH of about 4-10, more preferably about 5-9 or
about 6-8.5. fungal (e.g., yeast) cell culture media prepared by
the present methods will, upon reconstitution, preferably have a pH
of about 3-8, more preferably about 4-8 or about 4-7.5; animal cell
culture media prepared by the present methods will, upon
reconstitution, preferably have a pH of about 6-8 or about 7-8,
more preferably about 7-7.5 or about 7.2-7.4; and plant cell
culture media prepared by the present methods will, upon
reconstitution, preferably have a pH of about 4-8, preferably about
4.5-7, 5-6 or 5.5-6. Of course, optimal pH for a given culture
medium to be used on a particular cell type may also be determined
empirically by one of ordinary skill using art-known methods.
[0146] In another example, one or more buffer salts, e.g., sodium
bicarbonate, may be added directly to a powdered nutritive medium
by agglomerating the buffer(s) into the medium using a fluid bed
apparatus, or by spray-drying the buffer(s) onto a dry or
agglomerated powdered medium (using a spray-drying apparatus as
described below). In a related aspect, a pH-adjusting agent such as
an acid (e.g., HCl) or a base (e.g., NaOH) may be added to a
powdered nutritive medium, which may contain one or more buffer
salts (such as sodium bicarbonate), by agglomeration of the
pH-adjusting agent into the powdered nutritive medium in a fluid
bed apparatus, by spray-drying the pH-adjusting agent onto the
powdered or agglomerated nutritive medium, or by a combination
thereof; this approach obviates the subsequent addition of a
pH-adjusting agent after reconstitution of the powdered medium.
Thus, the invention provides a powdered nutritive culture medium
useful in cultivation or growth of cells in vitro that, upon
reconstitution with a solvent (e.g., water or serum), has a pH that
is optimal for the support of cell cultivation or growth without a
need for adjustment of the pH of the liquid medium. This type of
medium, referred to herein interchangeably as "auto-pH" or
"automatically pH-adjusting" medium, therefore obviates the
time-consuming and error-prone steps of adding buffer(s) to the
medium after reconstitution and adjusting the pH of the medium
after dissolution of the buffer(s). For example, a mammalian cell
culture medium prepared according to these methods may, upon
reconstitution, have a pH of between about 7.1 to about 7.5, more
preferably between about 7.1 to about 7.4, and most preferably
about 7.2 to about 7.4 or about 7.2 to about 7.3.
[0147] In accordance with certain methods of the present invention,
automatically pH adjusting media can be produced by preparing
reconstituted media without the addition of any buffering systems
or pH-adjusting agents (an "auto-pH medium" of the invention). In a
preferred such aspect, an auto-pH medium maybe provided by
adjusting the buffering systems present in the medium. For example,
as one of ordinary skill is aware, culture media typically contain
buffers or buffering systems. By adjusting the pH-opposing forms of
such buffers in the medium, the invention provides for production
of an auto-pH medium, avoiding the requirement to add additional
buffers or pH-adjusting agents to achieve a proper pH level prior
to or upon reconstitution of the medium and prior to use. In one
such aspect of the inventon, pH-opposing forms of certain media
components (particularly phosphate or other buffer salts) are then
used in the culture medium to provide a desired pH upon
reconsitution of the powdered media. (pH-opposing forms of
components are conjugate acid-base pairs in which the members of
the pair can either raise the pH or lower it to achieve the desired
pH of the solution. Sodium HEPES (pH raising) and HEPES-HCl (pH
lowering) are examples ofpH opposing components.) For example, if a
reconstituted media having a pH of between 4.5 and 7.2 is to be
prepared, the first step is to determine the correct balance of
monobasic (to lower the pH) to dibasic (to raise the pH) phosphate
in order to yield the desired pH. Typically, mono- and dibasic
phosphate salts are used at concentrations of about 0.1 mM to about
10 mM, about 0.2 mM to about 9 mM, about 0.3 mM to about 8.5 mM,
about 0.4 mM to about 8 mM, about 0.5 mM to about 7.5 mM, about 0.6
mM to about 7 mM, or preferably about 0.7 mM to about 7 mM. If
other buffer systems are used in the formulations, the proper ratio
or balance of the basic (typically sodium or monobasic) buffer salt
and the corresponding acidic (or pH-opposing; typically HCl or
dibasic) buffer salt is similarly determined to ensure that the
formulation will be at the desired final pH upon reconstitution
with a solvent. Because the actual phosphate molecular species that
is present in a solution is the same at a given pH whether the
basic (e.g., sodium or monobasic) or acidic (e.g., HCl or dibasic)
form is added, this adjustment would not be expected to impact
buffering capacity. Once an appropriate ratio of pH-opposing forms
of an appropriate buffer is determined, these components may be
added to the medium (for example, a dry powder medium) to provide a
culture medium that is of the appropriate pH level upon
reconstitution and prior to use (i.e., an auto-pH medium of the
invention). The preparation of one example of such an automatically
pH-adjusting culture medium is described in more detail below in
Examples 3, 6 and 17.
[0148] In a related aspect, the invention provides for methods of
preparing culture media in such a way as to prevent the interaction
of media components that adversely affect the stability,
solubility, structure and/or performance of the medium. In one such
preferred aspect, the methods of the invention prevent the adverse
interaction between buffering components that are present in the
culture medium. For example, such methods of the invention may be
used to prevent off-gassing in the culture medium, which is the
release of gas from one or more medium components upon storage of
the medium in dry form prior to use. In particular, these methods
of the invention may be used to prevent off-gassing of carbon
dioxide from the medium, typically resulting from liberation of
carbon dioxide from a bicarbonate (particularly sodium bicarbonate)
buffer used in the medium. Sodium bicarbonate is generally not
included in powdered media because, depending on the type of
phosphate buffer used in the media, significant amounts of carbon
dioxide gas may be generated by off-gassing of the sodium
bicarbonate, which may swell a sealed container of the medium to
the bursting point, thus reducing the storage stability of the
finished product. To minimize this undesirable condition, dibasic
sodium phosphate (Na.sub.2HPO.sub.4) instead of monobasic sodium
phosphate (NaH.sub.2PO.sub.4) may be used in the formulations
comprising sodium bicarbonate. However, if monobasic sodium
phosphate is used in the formulation of the medium, monobasic
potassium phosphate (KH.sub.2PO.sub.4) can be used instead, which
does not result in gas formation, and thus does not cause
pillowing, while having the identical buffering capabilities as
monobasic sodium phosphate. According to this aspect of the
invention, the ratio of monobasic to dibasic phosphate (or other
buffer) salts to be used (or present) in the culture medium is
determined, and then the monobasic sodium phosphate (or other
monobasic buffer salt) is replaced with equal molar amount of
monobasic potassium phosphate, to prevent off-gassing of carbon
dioxide from the sodium bicarbonate in the medium. Since the
buffering capacity of monobasic sodium phosphate is identical to
that of monobasic potassium phosphate, this replacement would not
be expected to affect the buffering system present in the medium.
Thus, the invention provides culture media that prevent the adverse
interaction between components of the medium, particularly
preventing off-gassing, while still providing for auto-pH forms of
the culture media. The preparation of one example of such an
automatically pH-adjusting culture medium where the components of
the medium have been adjusted to minimize or prevent off-gassing is
described in more detail in Example 17.
[0149] Hence, as one of ordinary skill will recognize from the
description provided herein, the present invention also provides
complete dry powder culture media formulations that support the
cultivation of cells in vitro upon reconstitution of the medium
with a solvent, without the need for the addition of any
supplemental nutrient components to the medium prior to use. Media
according to this aspect of the invention thus will preferably
comprise the nutritional components necessary for cultivation of a
cell in vitro, such that no additional nutritional components need
be included in the solvent or added to the medium upon
reconstitution and prior to use. Accordingly, such complete media
of the invention will be suitable for use in cultivating cells in
vitro upon reconstitution with water or with an alternative
non-nutrient-containing solvent such as a buffered saline solution.
In accordance with the invention, such complete media may be
automatically pH-adjusting media, and may comprise one or more
components such one or more culture medium supplements (including
but not limited to serum), one or more amino acids (including but
not limited to L-glutamine), insulin, transferrin, one or more
hormones, one or more lipids, one or more growth factors, one or
more cytokines, one or more neurotransmitters, one or more extracts
of animal tissues, organs or glands, one or more enzymes, one or
more proteins, one or more trace elements, one or more
extracellular matrix components, one or more antibiotics, one or
more viral inhibitors, and or one or more buffers.
[0150] Examples of media supplements that may be prepared as
powders by the present methods, or that may be included in the
culture media of the invention, include, without limitation, animal
sera (such as bovine sera (e.g., fetal bovine, newborn calf and
calf sera), human sera, equine sera, porcine sera, monkey sera, ape
sera, rat sera, murine sera, rabbit sera, ovine sera and the like),
defined replacements such as LipoMAX.RTM., OptiMAb.RTM.,
Knock-Out.TM. SR (each available from Invitrogen Corporation,
Carlsbad, Calif.), hormones (including steroid hormones such as
corticosteroids, estrogens, androgens (e.g., testosterone) and
peptide hormones such as insulin, cytokines (including growth
factors (e.g., EGF, aFGF, bFGF, HGF, IGF-1, IGF-2, NGF and the
like), interleukins, colony-stimulating factors, interferons and
the like), neurotransmitters, lipids (including phospholipids,
sphingolipids, fatty acids, cholesterol and the like), attachment
factors (including extracellular matrix components such as
fibronectin, vitronectin, laminins, collagens, proteoglycans,
glycosaminoglycans and the like), and extracts of animal tissues,
organs or glands (such as bovine pituitary extract, bovine brain
extract, chick embryo extract, bovine embryo extract, chicken meat
extract, achilles tendon and extracts thereof) and the like). Other
media supplements that may be produced by the present methods or
that may be included in the culture media of the invention include
a variety of proteins (such as serum albumins, particularly bovine
or human serum albumins; immunoglobulins and fragments or complexes
thereof; aprotinin; hemoglobin; haemin or haematin; enzymes (such
as trypsin, collagenases, pancreatinin or dispase); lipoproteins;
fetuin; ferritin; etc.), which may be natural or recombinant;
vitamins; amino acids and variants thereof (including, but not
limited to, L-glutamine and cystine), enzyme co-factors;
polysaccharides; salts or ions (including trace elements such as
salts or ions of molybdenum, vanadium, cobalt, manganese, selenium,
and the like); and other supplements and compositions that are
useful in cultivating cells in vitro that will be familiar to one
of ordinary skill. These sera and other media supplements are
available commercially (for example, from Invitrogen Corporation,
Carlsbad, Calif., and Sigma Cell Culture, St. Louis, Mo.);
alternatively, sera and other media supplements described above may
be isolated from their natural sources or produced recombinantly by
art-known methods that will be routine to one of ordinary skill
(see Freshney, R. I., Culture of Animal Cells, New York: Alan R.
Liss, Inc., pp. 74-78 (1983), and references cited therein; see
also Harlow, E., and Lane, D., Antibodies: A Laboratory Manual,
Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, pp.
116-120 (1988)).
[0151] Components that are present in the final formulation in
.mu.g/ml or even .mu.g/L amounts have typically been left out of
standard powdered media due to homogeneity and/or stability
concerns, and instead are typically added to the reconstituted
1.times. media as a concentrate, thereby increasing storage costs
and causing production of a finished culture medium to become more
costly and less efficient. Thus, in one preferred aspect of the
present invention, such low-level components may be added to
standard powdered media by first making a concentrate of the
components and then spraying them into a portion of the powdered
media that would be granulated with the concentrate (See U.S.
application Ser. No. 09/023,790, filed Feb. 13, 1998, which is
incorporated herein by reference in its entirety). This would then
be milled (e.g., via Fitzmilling) to a particle size in the same
general size range as that of the bulk for blending. The ability to
spray-in components in small amounts maybe especially helpful in
developing media that include trace elements, vitamins, viral
inhibitors, growth factors, cytokines and the like. Specifically,
among others, the components to be added to a powdered medium
include but are not limited to calcium, choline chloride, folic
acid, inositol, lipoic acid, riboflavin, thiamine hydrochloride,
sodium selenite and vitamins A, B.sub.1, B.sub.2, B.sub.3, B.sub.6,
B.sub.12, C, D, E, K and H (biotin). Additional components to be
added in low amounts to the culture media of the invention may
include, for example, growth factors (e.g., EGF, aFGF, bFGF, KGF,
HGF, IGF-1, IGF-2, NGF, insulin, and the like), interleukins,
colony-stimulating factors, interferons, attachment factors,
extracellular matrix components (e.g., collagens, laminins,
proteoglycans, glysoaminoglycans, fibronectin, vitronectin, and the
like), lipids (such as phospholipids, cholesterol, bovine
cholesterol concentrate, fatty acids, sphingolipids and the like);
extracts of animal tissues, glands or organs; antibiotics such as
Geneticin.RTM., carbenicillin, cefotaxime, anti-PPLO,
Fungizone.RTM., hygromycin, kanamycin, neomycin, nystatin,
penicillin, or streptomycin, etc.; and viral inhibitors (e.g.,
protease inhibitors, nucleoside analogues, and the like, which are
well-known in the art).
[0152] Examples of buffers that may be prepared according to the
present invention and/or that may be included in the culture media
of the present invention include, but are not limited to, buffered
saline solutions such as phosphate-buffered saline (PBS)
formulations, Tris-buffered saline (TBS) formulations,
HEPES-buffered saline (HBS) formulations, Hanks Balanced Salt
Solutions (HBSS), Dulbecco's PBS (DPBS), Earle's Balanced Salt
Solutions, Puck's Saline Solutions, Murashige and Skoog Plant Basal
Salt Solutions, Keller's Marine Plant Basal Salt Solutions,
Provasoli's Marine Plant Basal Salt Solutions, Kao and Michayluk's
Basal Salt Solutions, and the like. Formulations for these buffers,
which are commercially available, as well as for many other
commonly used buffers, are well-known in the art and may be found
for example in the GIBCO/BRL Catalogue and Reference Guide
(Invitrogen Corporation, Carlsbad, Calif.), in the DEFCO Manual
(DIFCO; Norwood, Mass.), and in the Sigma Cell Culture Catalogues
for animal and plant cell culture (Sigma; St. Louis, Mo.).
Preparation of Powdered Media, Media Supplements, Media Subgroups
and Buffers
[0153] The methods of the present invention provide for the
preparation of the above-described powdered nutritive media, media
supplements, media subgroups and buffers. These powdered media,
supplements, subgroups and buffers are preferably prepared using
fluid bed technology (i.e., "agglomeration"), via tumble
granulation, and/or via spray-drying.
[0154] In one aspect of the invention, the powdered nutritive
media, media supplements, media subgroups and buffers are prepared
using fluid bed technology to agglomerate the solutions of media,
media supplements, media subgroups or buffers, thereby producing
their dry powdered forms. Fluid bed technology is a process of
producing agglomerated powders having altered characteristics
(particularly, for example, solubility) from the starting
materials. In general applications of the technology, powders are
suspended in an upwardly moving column of air while at the same
time a controlled and defined amount of liquid is injected into the
powder stream to produce a moistened state of the powder; mild heat
is then used to dry the material, producing an agglomerated
powder.
[0155] Apparatuses for producing and/or processing particulate
materials by fluid bed technology are available commercially (e.g.,
from Niro, Inc./Aeromatic-Fielder; Columbia, Md.), and are
described, for example, in U.S. Pat. Nos. 3,771,237; 4,885,848;
5,133,137; 5,357,688; and 5,392,531; and in WO 95/13867; the
disclosures of all of the foregoing patents and applications are
incorporated by reference herein in their entireties. Such
apparatuses have been used to prepare agglomerated powders of
various materials, including milk whey (U.S. Pat. No. 5,006,204),
acidulated meat emulsions (U.S. Pat. No. 4,511,592), proteases
(U.S. Pat. No. 4,689,297) and other proteins (DK 167090 B1), and
sodium bicarbonate (U.S. Pat. No. 5,325,606).
[0156] According to this aspect of the invention, fluid bed
technology may be used to prepare bulk agglomerated nutritive
media, media supplements, media subgroups and buffers. In the
practice of this aspect of the invention, a dry powdered nutritive
medium, medium supplement or buffer is placed into a fluid bed
apparatus and is subjected to agglomeration therein. Powdered
nutritive media (particularly powdered cell culture media),
powdered media supplements (particularly powdered animal sera) and
powdered buffers (particularly powdered buffered salines), may be
obtained pre-made from commercial sources (e.g., Invitrogen
Corporation, Carlsbad, Calif.). Alternatively, powdered nutritive
media, media supplements, media subgroups or buffers may be made by
admixing individual components or sets of components according to
the formulations described above. Such formulations may include
components which typically are not present in powdered nutritive
media, media supplement, media subgroup and buffer formulations due
to their instability, such as serum, L-glutamine, cystine, insulin,
transferrin, lipids (particularly phospholipids, sphingolipids,
fatty acids and cholesterol), cytokines (particularly growth
factors, interleukins, colony-stimulating factors and interferons),
neurotransmitters and buffers (particularly sodium bicarbonate). If
L-glutamine is added to the formulation, it may be in the form of a
complex with divalent cations such as calcium or magnesium (see
U.S. Pat. No. 5,474,931). In another example, two or more powdered
components may be admixed and then agglomerated to produce complex
media, media supplements, media subgroups or buffers. For example,
a powdered nutritive medium may be mixed with a powdered serum
(produced, for example, by spray-drying as described below) such as
FBS at a serum concentration of about 0.1 %, 0.2%, 0.5%, 1%, 2%,
2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 50% or higher (w/w as a
percentage of the powdered medium); the resulting powdered
medium-serum mixture may then be agglomerated to produce an
agglomerated medium-serum complex that will readily dissolve in a
reconstituting solvent and thus be ready for use without further
supplementation.
[0157] Once the powdered nutritive media, media supplement, media
subgroup or buffer (or mixture thereof) is placed into the fluid
bed apparatus, it is subjected to suspension in an upwardly moving
column of a gas, preferably atmospheric air or an inert gas such as
nitrogen, and is passed through one or more particle filters. Since
most dry powder, non-agglomerated nutritive media, media
supplements, media subgroups and buffers are of a relatively small
particle size, filters to be used in the invention should be mesh
screens that allow air to flow through but that retain the powders,
for example filters of about 1-100 mesh, preferably about 2-50
mesh, more preferably about 2.5-35 mesh, still more preferably
about 3-20 mesh or about 3.5-15 mesh, and most preferably about 4-6
mesh.
[0158] After placement within the fluid bed chamber, the dry powder
nutritive media, media supplement, media subgroup or buffer (or
mixture thereof) is then subjected to agglomeration by injecting,
preferably using a spray nozzle on the fluid bed apparatus, a
defined and controlled amount of solvent into the powder, to
produce a moistened powder. Preferred solvents for use in the
present invention are any solvent that is compatible with the
formulation of the nutritive media, media supplement, media
subgroup or buffer. By "compatible" is meant that the solvent does
not induce irreversible deleterious changes in the physical or
performance characteristics of the nutritive media, media
supplement, media subgroup or buffer, such as breakdown or
aggregation of the nutrient components of the nutritive medium or
changes in the ionic characteristics of the buffer. Particularly
preferred solvents for use in the invention are water (most
particularly distilled and/or deionized water), serum (particularly
bovine or human serum and most particularly fetal bovine serum or
calf serum), organic solvents (particularly dimethylsulfoxide,
acetone, ethanol and the like), any of which may contain one or
more additional components (e.g., salts, polysaccharides, ions,
detergents, stabilizers, etc.).
[0159] As noted above, in some aspects of the invention, it may be
desirable or advantageous to include in the solvent one or more
ingredients that, due to the low concentrations of the components
present in the final product, cannot be optimally incorporated into
the product by other methods such as ball-milling. In one such
aspect, such low-concentration components may be dissolved,
suspended, colloided or otherwise introduced into the solvent at
the desired concentration, prior to use of the solvent in
agglomeration of the powdered media, media supplement, media
subgroup or buffer of the invention. Components that may be
advantageously incorporated into the solvent in accordance with
this aspect of the invention include, but are not limited to, one
or more of the above-described sera, hormones, cytokines,
neurotransmitters, lipids, attachment factors, proteins, amino
acids, vitamins, enzyme cofactors, polysaccharides, salts, ions,
buffers and the like.
[0160] The solvent should be introduced into the dry powder in a
volume that is dependent upon the mass of powdered media, media
supplement, media subgroup or buffer to be agglomerated. Preferred
volumes of solvent per 500 grams of nutritive media, media
supplement, media subgroup or buffer are about 5-100 ml, more
preferably about 10-50 ml, still more preferably about 25-50 ml,
and most preferably about 35 ml. Preferred solvent introduction
rates per 500 grams of nutritive media, media supplement, media
subgroup or buffer are a rate of about 1-10 ml/min, preferably
about 2-8 ml/min, more preferably about 4-8 m/min and most
preferably about 6 ml/min. In some situations, it may be desirable
to cycle between adding solvent for about one minute and then not
adding solvent for about one minute (allowing drying of the powder
within the apparatus chamber), so as to prevent clumping of the
powder during agglomeration.
[0161] Once agglomeration of the powder is complete, as evidenced
by a larger particle size than that of the original, unagglomerated
powder and by the absence of fine dust particles in the
agglomerated powder, the powder is thoroughly dried in the
apparatus. Preferred apparatus temperatures for drying of the
agglomerated powder are about 50-80.degree. C., more preferably
about 55-75.degree. C., and most preferably about 60-65.degree. C.;
powder is preferably dried in the apparatus for about 3-10 minutes
and most preferably for about 5-7 minutes, per 500 grams of
powder.
[0162] In an alternative aspect, powdered nutritive media, media
supplements, media subgroups and buffers of the invention may be
prepared by tumble granulation, which produces an agglomerated
product analogous to that described above. In such a process, dry
powder media, media supplements, media subgroups and/or buffers, or
combinations thereof, are introduced into a tumble granulator or a
tumble blender such as those that are commercially available from
Gemco (Middlesex, N.J.) and Patterson Kelley (East Stroudsburg,
Pa.). A solvent (e.g., water, buffered saline, or other desirable
solvent that is described herein or that will be familiar to one of
ordinary skill) is then introduced into the powder under controlled
conditions according to manufacturer's specifications in the tumble
granulator, and the batch is then dried according to manufacturer's
specifications to form granulated powder (i.e., granules of powder
containing solvent), which may then be used as described herein for
agglomerated powders.
[0163] In another aspect of the invention, powdered nutritive
media, media supplements, media subgroups and buffers may be
prepared by spray-drying. In this aspect of the invention, the
nutritive media, media supplements, media subgroups and buffers in
their liquid forms are placed into a spray-drying apparatus; these
liquids are then converted into their corresponding powders by
spraying the solution into a chamber in the apparatus under
appropriate conditions to produce the powders, such as under
controlled temperature and humidity, until the powders are formed.
In some situations, it may be desirable or advantageous to
spray-dry complex mixtures of two or more of the above-described
media, media supplements, media subgroups and/or buffers. For
example, liquid nutritive media containing animal sera at a desired
concentration, or liquid animal sera containing nutritive media
components at desired concentrations, may be mixed and then
prepared as spray-dried powders according to the methods of the
invention.
[0164] In a typical spray-drying approach, the liquid nutritive
media, media supplements, media subgroups and buffers are aspirated
into the apparatus and are atomized into a spray with a rotary- or
nozzle-type atomizer. The resulting atomized liquid spray is then
mixed with a gas (e.g., nitrogen or more preferably air) and
sprayed into a drying chamber under conditions sufficient to
promote production of a powdered product. In a preferred aspect of
the invention, these conditions may comprise electronic control of
the temperature and humidity within the chamber such that final
drying of the product is promoted. Under these conditions, the
solvent in the liquid evaporates in a controlled manner, thereby
forming free-flowing particles (i.e., powder) of the nutritive
media, media supplements, media subgroups or buffers of the
invention. The powder is then discharged from the drying chamber,
passed through one or more filters (such as the mesh screens
described above for fluid bed preparation) and collected for
further processing (e.g., packaging, sterilization, etc.). In some
applications, particularly when producing powders from
heat-sensitive formulations of nutritive media, media supplements,
media subgroups and buffers, the spray-drying apparatus may be
combined with a fluid bed apparatus integrated within the drying
chamber, which allows the introduction of agglomerating solvents
such as those described above into the spray-dried powder to
produce agglomerated spray-dried powdered nutritive media, media
supplements, media subgroups and buffers.
[0165] Apparatuses for producing particulate materials from liquid
materials by spray-drying (with or without integrated fluid bed
technology) are available commercially (e.g., from Niro,
Inc./Aeromatic-Fielder; Columbia, Md.), and are described, for
example, in the "Spray Drying," "Powdered Pharmaceuticals by Spray
Drying" and "Fresh Options in Drying" technical brochures of Niro,
Inc./Aeromatic-Fielder, the disclosures of which are incorporated
by reference herein in their entireties. According to this
manufacturer, such apparatuses have been used to prepare powders of
various materials, including dairy products, analgesics,
antibiotics, vaccines, vitamins, yeasts, vegetable protein, eggs,
chemicals, food flavorings and the like. In the present invention,
spray-drying has been found to be particularly useful for the
preparation of powdered media supplements, such as sera and in
particular those sera described above, most particularly human and
bovine sera (such as fetal bovine serum and calf serum).
[0166] In the practice of this aspect of the invention, the liquid
nutritive media, media supplements, media subgroups, buffers or
pH-adjusting agents should be sprayed into the chamber through the
atomizer at a spray rate of about 25-100 g/min, preferably at a
spray rate of about 30-90 g/min, 35-85 g/min, 40-80 g/min, 45-75
g/min, 50-75 g/min, 55-70 g/min, or 60-65 g/min, and more
preferably at about 65 g/min. The inlet air temperature in the
atomizer is preferably set at about 100-300.degree. C., more
preferably at about 150-250.degree. C., and most preferably at
about 200.degree. C., with an outlet temperature of about
50-100.degree. C., more preferably about 60-80.degree. C., and most
preferably about 70.degree. C. Air flow in the atomizer is
preferably set at about 50-100 kg/hr, more preferably about 75-90
kg/hr, and most preferably about 80.0 kg/hr, at a nozzle pressure
of about 1-5 bar, more preferably about 2-3 bar, and most
preferably about 2.0 bar. These conditions and settings have been
found in the present invention to be preferable for production of a
variety of nutritive media, media supplements, media subgroups and
buffer powders by spray-drying, particularly for the production of
the above-described powdered sera. Following drying, the
spray-dried powdered nutritive media, media supplements, media
subgroups or buffers may be collected in the drying chamber through
one or more filters, preferably such as those described above for
fluid bed technology.
[0167] Following this preparation, the powders of the invention
prepared by the above-described fluid bed or spray-drying methods
have altered physical characteristics from the starting powders or
from powdered media, supplements, subgroups and buffers prepared by
lyophilizing the corresponding liquids. For example, non-processed
or lyophilized powders often produce significant dust when used,
and dissolve poorly or slowly in various solvents, while
agglomerated or spray-dried powders are substantially dust-free
and/or dissolve rapidly. Typically, the powdered media, media
supplements, media subgroups and buffers of the invention will
exhibit both reduced dusting and more rapid dissolution than their
powdered counterparts prepared by standard techniques such as
ball-milling. In some powders which are substantially dust-free but
which may not demonstrate enhanced dissolution, the powders maybe
rapidly dissolved by rapid mechanical solvation of the powder, such
as using a mechanical impeller, or by first providing a solvent
mist over the powder such as by spray solvation.
[0168] In another aspect of the invention, the spray-drying and
agglomeration approaches described above may be combined to produce
agglomerated spray-dried nutritive media, media supplement, media
subgroup and buffer powders. In this aspect, a powdered medium,
supplement, subgroup or buffer that has been prepared by
spray-drying may, after having been spray-dried, then be
agglomerated with a solvent (such as those described above) to
further improve the performance and physical characteristics of the
resultant medium, supplement, subgroup or buffer. For example, an
animal serum powder may be prepared by spray-drying liquid animal
serum as described above, and this spray-dried serum powder may
then be mixed into dry powder nutritive media (prepared by
spray-drying or by standard techniques such as ball-milling); this
mixed powder may then be agglomerated as described above.
Alternatively, a spray-dried nutritive medium, medium supplement,
medium subgroup or buffer powder may be agglomerated as above, to
improve the dissolution properties of the powder. This approach may
be particularly advantageous when spray-drying liquids with low
(about 1-10%) solids content, such as liquid animal sera. As one of
ordinary skill will appreciate, these approaches will facilitate
preparation of a large batch of one or more components (e.g., sera
or other media supplements) to be used as a stock for addition to a
powdered medium, supplement, subgroup or buffer at a desired
concentration, while also obtaining the above-described benefits of
agglomeration. In addition, this approach may reduce inter-lot
variability which may be a problem with certain media supplements
(particularly animal sera).
[0169] The powdered nutritive media, media supplements, media
subgroups or buffers prepared as described herein may then be
packaged, for example into containers such as vials, tubes,
bottles, bags, pouches, boxes, cartons, drums and the like, prior
to or following sterilization as described below. In one such
aspect of the invention, the powdered media, media supplements,
media subgroups or buffers may be packaged into a compact,
vacuum-packed form, such as that known in the art as a "brick-pack"
wherein the powder is packaged into a flexible container (such as a
bag or a pouch) that is sealed while being evacuated. Such packages
may advantageously comprise one or more access ports (such as
valves, luer-lock ports, etc.) allowing the introduction of a
solvent (e.g., water, sera, media or other aqueous or organic
solvents or solutions) directly into the package to facilitate
rapid dissolution of the powder. In a related aspect, the package
may comprise two or more adjacent compartments, one or more of
which may contain one or more of the dry powder media, media
supplements, media subgroups or buffers of the invention and one or
more other of which may contain one or more aqueous or organic
solvents which may be sterile. In this aspect, the dry powder may
then be dissolved by simply removing or breaking the barrier
between the compartments, ideally without loss of sterility, to
allow admixture of the powder and the solvent such that the powder
dissolves and produces a sterile nutritive medium, medium
supplement, medium subgroup or buffer at a desired
concentration.
[0170] Packaged media, media supplements, media subgroups and
buffers of the invention are preferably stored for the extended
times, and at the temperatures, noted above, typically for about
1-24 months at temperatures of less than about 30.degree. C., more
preferably at temperatures of less than about 20-25.degree. C.,
until use. Unlike traditional powdered media, media supplements,
media subgroups or buffers, storage at reduced temperatures (e.g.,
0-4.degree. C.) is not necessary for the maintenance of performance
characteristics of the media, media supplements, media subgroups
and buffers prepared by the present methods. Of course, other
storage temperatures may be required for those aspects of the
invention where the packages also comprise separate compartments
containing one or more solvents; in these cases, the optimal
storage conditions will be dictated by the storage requirements of
the solvent(s) which will be known to the skilled artisan.
Sterilization and Packaging
[0171] The invention also provides methods for sterilizing the
nutritive media, media supplements, media subgroups and buffers of
the invention, as well as for sterilizing powdered nutritive media,
media supplements, media subgroups and buffers prepared by standard
methods such as ball-milling or lyophilization. Since nutritive
media, media supplements, media subgroups and buffers are usually
prepared in large volume solutions and frequently contain heat
labile components, they are not amenable to sterilization by
irradiation or by heating. Thus, nutritive media, media
supplements, media subgroups and buffers are commonly sterilized by
contaminant-removal methods such as filtration, which significantly
increases the expense and time required to manufacture such media,
media supplements, media subgroups and buffers.
[0172] Powdered nutritive media, media supplements, media subgroups
and buffers prepared according to the methods of the invention
(e.g., by spray-drying of liquid media, media supplements, media
subgroups or buffers, or by agglomeration of powdered media, media
supplements, media subgroups or buffers), or by standard methods
such as ball-milling (of powdered components) or lyophilization (of
liquid forms of the media, supplements, subgroups or buffers),
however, can be sterilized by less expensive and more efficient
methods. For example, powdered nutritive media, media supplements,
media subgroups or buffers (prepared as described above by
spray-drying or lyophilization of a liquid form, or by
agglomeration of a powdered form, of the media, supplements,
subgroups or buffers) may be irradiated under conditions favoring
sterilization of these powders. Preferably, this irradiation is
accomplished in bulk (i.e., following packaging of the nutritive
media, media supplement, media subgroup or buffer), and most
preferably this irradiation is accomplished by exposure of the bulk
packaged media, media supplement, media subgroup or buffer of the
invention to a source of gamma rays under conditions such that
bacteria, fungi, spores or viruses that may be resident in the
powdered media, media supplements, media subgroups or buffers are
inactivated (i.e., prevented from replicating). Alternatively,
irradiation may be accomplished by exposure of the powdered media,
media supplement, media subgroup or buffer, prior to packaging, to
a source of gamma rays or a source of ultraviolet light. The media,
media supplements, media subgroups and buffers of the invention may
alternatively be sterilized by heat treatment (if the subgroups of
the nutritive media, media supplement, media subgroup or buffer are
heat stable), for example by flash pasteurization or autoclaving.
As will be understood by one of ordinary skill in the art, the dose
of irradiation or heat, and the time of exposure, required for
sterilization will depend upon the bulk of the materials to be
sterilized, and can easily be determined by the ordinarily skilled
artisan without undue experimentation using art-known techniques,
such as those described herein.
[0173] In a particularly preferred aspect of the invention, the
bulk powdered nutritive media, media supplements, media subgroups
or buffers are exposed to a source of .gamma. irradiation at a
total dosage of about 10-100 kilograys (kGy), preferably a total
dosage of about 15-75 kGy, 15-50 kGy, 15-40 kGy or 20-40 kGy, more
preferably a total dosage of about 20-30 kGy, and most preferably a
total dosage of about 25 kGy, for about 1 hour to about 7 days,
more preferably about 1 hour to about 5 days, 1 hour to about 3
days, about 1-24 hours or about 1-5 hours, and most preferably
about 1-3 hours ("normal dose rate"). Alternatively, the bulk
powders of the invention may be sterilized at a "slow dose rate" of
a total dosage of about 25-100 kGy over a period of about 1-5 days.
During irradiation, the powdered nutritive media, media
supplements, media subgroups or buffers are preferably stored at a
temperature of about -70.degree. C. to about room temperature
(about 20-25.degree. C.), most preferably at about -70.degree. C.
One of ordinary skill will appreciate, of course, that radiation
dose and exposure times may be adjusted depending upon the bulk
and/or mass of material to be irradiated; typical optimal
irradiation dosages, exposure times and storage temperatures
required for sterilization of bulk powdered materials by
irradiation or heat treatment are well-known in the art.
[0174] Following sterilization, unpackaged nutritive media, media
supplements, media subgroups and buffers may be packaged under
aseptic conditions, for example by packaging the media, media
supplements, media subgroups or buffers into containers such as
sterile tubes, vials, bottles, bags, pouches, boxes, cartons, drums
and the like, or in the vacuum packaging or integrated
powder/solvent packaging described above. Sterile packaged media,
media supplements, media subgroups and buffers may then be stored
for extended periods of time as described above.
Use of the Nutritive Media, Media Supplements, Media Subgroups and
Buffers
[0175] The present invention thus provides powdered nutritive
media, media supplements, media subgroups and buffers that are
readily soluble in a rehydrating solvent and that are substantially
dust free. For use, the agglomerated or spray-dried media, media
supplement, media subgroup or buffer may be hydrated (or
"reconstituted") in a volume of a solvent sufficient to produce the
desired nutrient, electrolyte, ionic and pH conditions required for
the particular use of the solvated media, media supplement, media
subgroup or buffer. This reconstitution is particularly facilitated
in the present invention, since the present media, media
supplements, media subgroups and buffers will rapidly go into
solution and will produce little if any dust or insoluble material,
unlike lyophilized or ball-milled nutritive media, media
supplements, media subgroups or buffers.
[0176] Preferred solvents for use in reconstituting the powdered
nutritive media, media supplements, media subgroups and buffers of
the invention include, but are not limited to, water (most
particularly distilled and/or deionized water), serum (particularly
bovine or human serum and most particularly fetal bovine serum or
calf serum), organic solvents (particularly dimethylsulfoxide,
acetone, ethanol and the like), or any combination thereof, any of
which may contain one or more additional components (e.g., salts,
polysaccharides, ions, detergents, stabilizers, etc.). For example,
powdered media supplements (such as animal sera) and buffers are
preferably reconstituted in water to a 1.times. final
concentration, or optionally to a higher concentration (e.g.,
2.times., 2.5.times., 5.times., 10.times., 20.times., 25.times.,
50.times., 100.times., 500.times., 1000.times., etc.) for the
preparation of stock solutions or for storage. Alternatively,
powdered culture media may be reconstituted in a solution of media
supplements (e.g., sera such as FBS) in water, such as those
solutions wherein the media supplement is present at a
concentration, for example, of 0.5%, 1%, 2%, 2.5%, 5%, 7.5%, 10%,
15%, 20%, 25%, 50%, or higher, vol/vol in the water.
[0177] Reconstitution of the powdered nutritive media, media
supplements, media subgroups or buffers is preferably accomplished
under aseptic conditions to maintain the sterility of the
reconstituted media, media supplement, media subgroup or buffer,
although the reconstituted media, media supplement, media subgroup
or buffer may alternatively be sterilized, preferably by filtration
or other sterilization methods that are well-known in the art,
following rehydration. Following their reconstitution, media, media
supplements, media subgroups and buffers should be stored at
temperatures below about 10.degree. C., preferably at temperatures
of about 0-4.degree. C., until use.
[0178] The reconstituted nutritive media, media supplements, media
subgroups and buffers may be used to culture cells according to
standard cell culture techniques which are well-known to one of
ordinary skill in the art. In such techniques, the cells to be
cultured are contacted with the reconstituted media, media
supplement, media subgroup or buffer of the invention under
conditions favoring the cultivation of the cells (such as
controlled temperature, humidity, lighting and atmospheric
conditions). Cells which are particularly amenable to cultivation
by such methods include, but are not limited to, bacterial cells,
yeast cells, plant cells and animal cells. Such bacterial cells,
yeast cells, plant cells and animal cells are available
commercially from known culture depositories, e.g., American Type
Culture Collection (Rockville, Md.), Invitrogen (La Jolla, Calif.)
and others that will be familiar to one of ordinary skill in the
art. Preferred animal cells for cultivation by these methods
include, but are not limited to, insect cells (most preferably
Drosophila cells, Spodoptera cells and Trichoplusa cells), nematode
cells (most preferably C. elegans cells) and mammalian cells
(including but not limited to CHO cells, COS cells, VERO cells, BHK
cells, AE-1 cells, SP2/0 cells, L5.1 cells, hybridoma cells and
most preferably human cells such as 293 cells, PER-C6 cells and
HeLa cells), any of which may be a somatic cell, a germ cell, a
normal cell, a diseased cell, a transformed cell, a mutant cell, a
stem cell, a precursor cell or an embryonic cell, and any of which
may be an anchorage-dependent or anchorage-independent (i. e.,
"suspension") cell.
1. Supplements
[0179] Another aspect of the present invention features a dry
format media supplement comprising at least two powder components.
The preferred supplement is reconstitutable for addition to a cell
culture medium. The preferred medium is capable of supporting cell
growth and/or expansion and/or production biomolecules.
[0180] A preferred dry media supplement comprises a mixture of at
least one first component selected from the group consisting of an
amino acid or salt thereof, a polysaccharide, a solubilizing agent,
a vitamin, a lipid, a fatty acid, a hormone, a growth factor, a
differentiation factor, an active polypeptide, an iron chelator, a
divalent metal salt, a carbon source, a monovalent metal salt, a pH
buffer, a polyanion, a polycation, a surfactant, an antioxidant, a
trace element salt, a nucleotide, a heterocyclic base and a
nucleoside; and at least one second component having a different
chemical constitution than said first component, said second
component selected from the group consisting of an amino acid or
salt thereof, a polysaccharide, a solubilizing agent, a vitamin, a
lipid, a fatty acid, a hormone, a growth factor, a differentiation
factor, an active polypeptide, an iron chelator, a divalent metal
salt, a carbon source, a monovalent metal salt, a pH buffer, a
polyanion, a polycation, a surfactant, an antioxidant, a trace
element salt, a nucleotide, a heterocyclic base and a
nucleoside.
[0181] Another preferred supplement of the invention features a
supplement ent comprising at least one amino acid or salt thereof
selected from the group consisting of alanine, cysteine, cystine,
aspartic acid, glutaminc acid, phenylalanine, glycine, histidine,
isoleucine, lysine, leucine, methionine, asparagine, proline,
hydroxyproline, glutamine, arginine, serine, threonine, valine,
tryptophan and tyrosine.
[0182] Another preferred aspect of the invention features a
supplement of the present invention reconstituted in a polar
solvent, preferably reconstituted in essentially physiologic pH
water.
[0183] Yet another preferred supplement of the present invention
comprises at least one component selected from the group consisting
of alanine, cysteine, cystine, aspartic acid, glutaminc acid,
phenylalanine, glycine, histidine, isoleucine, lysine, leucine,
methionine, asparagine, proline, hydroxyproline, glutamine,
arginine, serine, threonine, valine, tryptophan and tyrosine, in
free or salt form.
[0184] One or more amino acids, preferably 2, 3, 4, 5 or more amino
acids comprise a preferred aspect of supplements of the invention.
Salts of amino acids may be substituted for amino acisds in some
aspects of the present invention. Supplements containing at least
one amino acid and at least one vitamin are yet another aspect of
the invention.
[0185] Preferred powder preparation methods include one or more of
milling, impacting, extruding and cutting or breaking, wet
granulation, high shear granulation, pan granulation and fluidized
bed agglomeration.
[0186] Preferred milling apparatus include ball mill, roll mill,
fitz mill, comill, jet mill and hammer mill, and any other
mechanical particle size attrition device.
[0187] A preferred dry supplement formulation comprises an amino
acid or salt thereof, a vitamin, and a carbon source. Yet another
preferred formulation comprises at least one, possibly at least
two, preferably three, four five or more ingredients. Exemplary
ingredients may be selected from the group consisting of a metal
chelator, lipids, fatty acids, a divalent metal salt, a pH buffer,
and a carbon source. Other examples may be selected from the group
consisting of a polyanion, a polycation, a surfactant, an
antioxidant, a carbon source, a trace metal element salt, a
nucleotide, a heterocyclic base and a nucleoside.
[0188] Yet another aspect of the invention features a method of
reconstituting a dry medium supplement powder comprising obtaining
a powder according to the present invention and dissolving the
powder in essentially neutral pH water and or polar solvent.
[0189] An improvement in the art provided by the present invention
can be described as follows: a dry powder medium supplement for
supplementing cells growing in culture, the improvement being that
the supplement powder reconstitutes in essentially neutral pH water
and or polar solvent. Preferably, the reconstitution is rapid,
preferably less than five minutes per liter, more preferably less
than 4, 3, 2, or one minute per liter. Especially for large batch
sizes rapid easy reconstitution is preferred, for example, less
that 45, 30, 15t or even 10 seconds per liter.
[0190] A second improvement for some aspects is that pH adjustment
is not required.
[0191] A method of culturing cells comprising: growing cells in
culture for at least 2days to form a mature culture; reconstituting
a supplement powder using essentially neutral pH water and or polar
solvent to form a reconstituted supplement; and adding said
reconstituted supplement to said mature culture. A mature culture
can be a culture at a desired stage of growth, for example, a
culture adjudged to be ready for commencing bioproduction. A
preferred aspect provides that growth/expression/production of said
cells in culture is increased from that of the mature culture.
[0192] Another aspect of the invention is in kit form comprising at
least two containers of different composition of components wherein
at least one container contains a mixture of at least two
components. Preferably reconstitution of the components of at least
one container is in a polar solvent and/or the reconstitution of
the components of at least a second container is in essentially
physiologic pH water.
[0193] Preferred mixtures of the present invention are homogeneous
mixtures.
[0194] The method according to claim 22 wherein the pressure feed
is provided by a pump selected from the group consisting radial
flow, rotary, axial flow, regenerative, turbine, plunger,
diaphragm, cam and piston, peristaltic, gear, lobe, piston, screw,
syringe, metering, sliding-vane, hydraulic, jet, volumetric
displacement, and other reciprocating or positive displacement
pumps.
[0195] A preferred adding rate may range from about 1%/day to about
500%/day. Supplementation can be selected for ease of the additions
and results obtained. A tradeoff may be found in many aspects. For
example a preferred supplementation schedule may be once a day,
twice a day 3, 4, 5, 6, 7, or eight times per day, once every
several days, for example every two, two and a half, three, 5 or
even seven days. Supplementation may also be effected in accordance
with a culture monitoring system, wherein threshold values or one
or more algorithms are used to determine the feeding schedule.
Preferred percentages of supplementation include 1% per addition,
2, 3, 5, 7, 10, 20, 50%, continuous (perfusion) preferred rates
include 1 volume per day ranging from 25% to 5 volumes per day.
Preferably the adding is metered according to a feed back circuit
based on at least one measured concentration or size from a
component in the medium. Preferred monitoring includes cell volume,
number, size, concentration of glucose, any amino acid, a metabolic
product or metabolite, active oxygen, active oxygen products,
lactate, pH, Na, K, Ca, Se, viscosity, light absorbance, color
change, protein.
[0196] A preferred culture is one wherein the active polypeptide is
selected from the group consisting of an enzyme, a transport
protein, a membrane stabilizer, a neurotransmitter, a
differentiating agent and a binding or sequestering agent.
[0197] Another preferred medium supplement powder is one wherein
the vitamin is selected from the group consisting of ascorbate or
ascorbic acid or salt thereof, biotin, pantothenate or pantothenic
acid, bitartrate, choline chloride, cyanocobalamin, D- or DL-alpha
tocopherol or tocopherol acetate, folic acid, folinic acid or a
salt thereof, i-inositol, carnitine, lipoic acid, linoleic acid,
menadione or salt thereof, niacinamide, nicotinic acid,
para-aminobenzoic acid, pyridoxal 5-phosphate, pyidoxal HCl,
pyridoxamine mono or di hydrochloride, retinoic acid, riboflavin,
riboflavin-5-phosphate sodium dihydrate, thiamine HCl, Thiamine
monophosphate, vitamin A or salt thereof, Vitamin D2 and vitamin
D3.
[0198] A preferred supplement powder is one wherein the at least
two powder components are milled to produce a mean diameter ratio
of a largest component to a smallest component of 1:1 to 10:1.
[0199] Yet another preferred aspect features a powder supplement
wherein the at least two powder components are blended using tumble
blender including but not limited to drum blender, V-blender,
ribbon blender, cone blender, slant-cone blender and double-cone
blender sufficient mixing time and speed to attain a homogenous
mixture of components.
[0200] The present invention also features method of producing a
medium supplement, said method comprising obtaining a dry format
media supplement said supplement comprising at least two powder
components and said supplement reconstitutable for addition to a
cell culture medium, said medium capable of supporting cell growth,
biomolecule production and/or expansion; and dissolving said
supplement in a solvent to produce said medium supplement; and
optionally adding said medium supplement to said medium.
[0201] A preferred method is one wherein at least one of said at
least two components is present at a concentration in excess of a
1.times. concentration with respect to the medium being
supplemented. A more preferred method is one wherein at least one
of said at least two components is present at a concentration of
from greater than a 1.times. to about a 5.times., 10.times.,
20.times., 50.times., 75.times., 100.times., 120.times.,
150.times., 200.times., 250.times., 500.times., 750.times. or up to
a 1000.times. concentration with respect to the medium being
supplemented.
[0202] An especially preferred method is one wherein at least one
of said at least two components is present at a concentration in
excess of about 4.times. concentration with respect to the medium
being supplemented.
[0203] Another especially preferred method is one wherein at least
one of said at least two components is present at a concentration
in excess of about a 9.times. concentration with respect to the
medium being supplemented. Yet another preferred method is one
wherein at least one of said at least two components is present at
a concentration of about 5.times. with respect to the medium being
supplemented. Yet still another preferred method is one wherein at
least one of said at least two components is present at a
concentration of about 10.times. with respect to the medium being
supplemented. Perhaps two or more, for example, 3, 4, 5, 6, 7, 8,
9, or more components including essentially all components of the
supplement powder may reach any of these concentrations before
addition.
[0204] Another aspect of the invention is a powder wherein a first
component of the medium being supplemented is omitted or is present
at a concentration with respect to said medium being supplemented
in comparison to a second component with respect to said medium
being supplemented at a ratio greater than 0, range 10-6 to 0.9;
Exemplary supplements are those wherein said first component is
selected from the group consisting of alanine, cysteine, cystine,
aspartic acid, glutaminc acid, phenylalanine, glycine, histidine,
isoleucine, lysine, leucine, methionine, asparagine, proline,
hydroxyproline, glutamine, arginine, serine, threonine, valine,
tryptophan and tyrosine, an alkali metal salt, a pH buffer and a
surfactant.
[0205] Especially preferred powders include those wherein all
components are soluble in essentially physiologic pH water at a
concentration of from about 2.times., about 4.times., about
5.times., about 10.times., about 15.times., about 20.times., about
25.times., about 30.times., about 40.times., about 45.times., to
about 48.times. or about 50.times. or from about 50.times. to about
100.times., about 120.times., about 150.times., about 180.times.,
about 190.times. or about 200.times. of the medium being
supplemented.
[0206] Preferably all supplement components are of non-animal
origin. More preferably the supplement is chemically defined.
[0207] Another aspect is one wherein no component is serum or a
product of serum.
[0208] A preferred powder format is one that is prepared by
fluidized bed agglomeration.
[0209] Preferably supplement powders comprise at least one sugar.
Another preferred dry format powder supplement comprises a
polymeric binder such as methyl cellulose and derivative of starch,
ethyl cellulose and polyvinylpyrrolidone (PVP).
[0210] Another dry format media supplement is one wherein at least
a first powder component is designated as 1.times. per unit weight
based on the medium to be supplemented and a second powder
component is greater than 1.4.times. or less than 0.8.times. per
said unit weight.
[0211] Another preferred supplement may be one wherein at least
said second powder component is not present in said medium to be
supplemented.
[0212] Preferably upon reconstitution with a solvent a result is
achieved wherein in at least one component concentration greater
than about 2.times. that of said medium component
concentration.
[0213] A preferred supplement is one wherein at least one component
is soluble at at least 2.times. in physiologic pH water.
[0214] Preferred supplements may comprise bicarbonate. Preferably
the bicarbonate is a salt of a monovalent metal.
[0215] A preferred supplement comprises at least one ingredient
selected from the group consisting of trace element salt.
Preferrably the one or more trace element salts are homogenously
distributed throughout the dry format media supplement.
[0216] A preferred result of the products and methods of the
present invention is one wherein said culturing results in creation
of or an increased production of a desired cell component, product
or function.
Cells
[0217] In another aspect, the invention relates to methods for
producing dry cell powder compositions comprising one or more
cells, and to dry cell powders produced by these methods. These
methods thus produce cell-containing compositions wherein the cells
are preserved and may be stored for extended periods of time until
use. In this way, the methods of the invention overcome some of the
drawbacks of traditional methods of cell preservation (e.g.,
freezing) such as the need for cyropreservation equipment and the
use of certain cryopreservatives that may be toxic to the
cells.
[0218] Methods according to this aspect of the invention may
comprise one or more steps. For example, one such method may
comprise obtaining one or more cells to be dried, forming an
aqueous cell suspension by suspending the one or more cells in an
aqueous solution, and spray-drying the cell suspension under
conditions favoring the production of a dried powder. These methods
may further comprise contacting the one or more cells with one or
more stabilizing or preserving compounds (e.g., a polysaccharide,
including but not limited to trehalose). The aqueous solution used
to form the cell suspension preferably comprises one or more
components, such as one or more of the above-described nutritive
media, media supplements, media subgroups, salts or buffers, and
particularly one or more of the automatically pH-adjusting culture
media, media subgroups, media supplements or buffers of the present
invention. Preferably, the aqueous solution used to form the cell
suspension is adjusted to optimal or substantially optimal tonicity
and osmolality for the cell type being dried. The aqueous solution
may optionally comprise one or more additional components, such as
one or more polysaccharides, ions, detergents, stabilizing or
preserving compounds (including trehalose), and the like. In
aspects of the invention wherein the one or more cells are
contacted with one or more stabilizing or preserving compounds, the
stabilizing or preserving compounds may be incorporated into the
aqueous solution used to form the aqueous cell suspension.
Alternatively, the stabilizing or preserving compounds may be
sprayed or agglomerated onto the dry cell powder after formation of
the powder.
[0219] Once the dry cell powder has been formed by the
above-described methods, the powder may optionally be agglomerated
with a solvent according to methods described above for
agglomeration of dry powders. Any solvent that is compatible with
the cell type being dried may be used to agglomerate the dry cell
powder, including but not limited to water, a nutritive medium
solution, a nutritive medium supplement solution (including sera,
particularly bovine sera (most particularly fetal bovine and calf
sera) and human sera), a buffer solution, a salt solution, and
combinations thereof.
[0220] A variety of cells maybe dried according to the methods of
the invention, including prokaryotic (e.g., bacterial) and
eukaryotic (e.g., fungal (especially yeast), animal (especially
mammalian, including human) and plant) cells, particularly those
cells, tissues, organs, organ systems, and organisms described
above. Once the dried cells have been produced, they may be
packaged aseptically and stored for extended periods of time (e.g.,
several months to several years), preferably at temperatures of
about 0-30.degree. C., 4-25.degree. C., 10-25.degree. C., or
20-25.degree. C. (i.e., "room temperature") until use. For use in
preparing cultures of viable cells, the dry cell powder may be
aseptically reconstituted, into a cell suspension comprising one or
more viable cells, with an aqueous solvent (e.g., sterile water,
buffer solutions, media supplements, culture media, or combinations
thereof) and cultured according to standard art-known protocols.
Alternatively, the dry cell powder may be reconstituted into a cell
suspension where cell viability is not essential, for example for
preparation of an immunogen to be used for immunization of an
animal. In such cases, the dry cell powder may be reconstituted
with any solvent that is compatible with standard immunization
protocols, such as aqueous or organic solvents that may comprise
one or more detergents, adjuvants, etc.
[0221] The invention also provides compositions prepared by such
methods. Such compositions may comprise, for example, an
automatically pH-adjusting culture medium powder of the invention
and one or more cells, such as one or more bacterial cells, one or
more plant cells, one or more yeast cells, and one or more animal
cells (including but not limited to one or more mammalian cells
such as one or more human cells). Compositions according to this
aspect of the invention may be in powder form which, upon
reconstitution with a solvent, produce an active culture of the one
or more cells contained in the composition.
Kits
[0222] The dry powder media, media supplements, media subgroups,
buffers, cells and cell-containing compositions provided by the
invention are ideally suited for preparation of kits. Such a kit
may comprise one or more containers such as vials, test tubes,
bottles, packages, pouches, drums, and the like. Each of the
containers may contain one or more of the above-described nutritive
media, media supplements, media subgroups or buffers of the
invention, or combinations thereof. Such nutritive media, media
supplements, media subgroups or buffers may be hydrated or
dehydrated but are typically dehydrated preparations produced by
the methods of the invention. Such preparations may, according to
the invention, be sterile.
[0223] A first container may contain, for example, a nutritive
media, media supplement, media subgroup or a buffer of the
invention, or any component or subgroup thereof, such as any of
those nutritive media, media supplements, media subgroups or
buffers of the invention that are described above. Additional
nutritive media, buffers, extracts, supplements, components or
subgroups may be contained in additional containers in the present
kits. The kits may also contain, in one or more additional
containers, one or more cells such as the above-described bacterial
cells, yeast cells, plant cells or animal cells. Such cells maybe
lyophilized, dried, frozen or otherwise preserved, or maybe
spray-dried according to the methods of the invention. In addition,
the kits of the invention may further comprise one or more
additional containers, containing, for example, L-glutamine,
optionally complexed with one or more divalent cations (see U.S.
Pat. No. 5,474,931). The kits may further comprise one or more
additional containers containing a solvent to be used in
reconstituting the dry powder nutritive media, media supplements,
media subgroups and/or buffers; such solvents may be aqueous
(including buffer solutions, saline solutions, nutritive medium
solutions, nutritive medium supplement solutions (including sera
such as bovine sera (particularly fetal bovine sera or calf sera)
or human sera), or combinations thereof) or organic. Other
ingredients that are not compatible for admixture with the
nutritive media, buffers, extracts, supplements, components or
subgroups of the invention may be contained in one or more
additional containers to avoid mixing of incompatible
components.
[0224] The number and types of containers contained in a given kit
for making a nutritive media, media supplement, media subgroup or
buffer may vary depending on the type of media, media supplement,
media subgroup or buffer to be prepared. Typically, the kit will
contain the respective containers containing the components or
supplements necessary to make a particular media, media supplement,
media subgroup or buffer. However, additional containers may be
included in the kit of the invention so that different media, media
supplements, media subgroups or buffers can be prepared by mixing
different amounts of various components, supplements, subgroups,
buffers, solvents, etc., to make different media, media supplement,
media subgroup or buffer formulations.
Advantages
[0225] Unexpectedly, the present invention provides for the
preparation of nutritive media, media supplements, media subgroups,
buffers and cells at reduced cost. The cost reductions are due to
the several factors. For example, the media, media supplement,
media subgroup and buffer formulations of the present invention may
be produced with much smaller production facilities since the large
stir tanks required for 1.times. formulations are not required. In
addition, the media, media supplement, media subgroup and buffer
formulations of the present invention may be prepared on an as
needed basis using "just in time" production techniques which
reduce inventory, storage and labor costs. The time required for
the preparation and shipping of the media, media supplement, media
subgroup and buffer formulations may be reduced from 6-8 weeks to
as little as one day. The automatically pH-adjusting media of the
invention also provide significant cost and time savings, and
reduce the tendency for introduction of contamination into
reconstituted media that may occur during the pH adjustment process
according to standard methods using traditional dry powder or bulk
liquid media. The present invention also allows for the preparation
of components of nutritive media, media supplements, media
subgroups or buffers which may be used to prepare very large
quantities of 1.times. media, media supplements, media subgroups or
buffers (e.g., 100,000 liters or more) which would require only one
quality control test compared to multiple quality control tests for
multiple batches produced according to other commonly used
techniques. Importantly, the media, media supplement, media
subgroup and buffer formulations of the present invention are more
consistent between batches since the individual components are more
stable. The dried cell powders of the invention are also
technologically and economically advantageous, since the cells
maybe stored, in low volume, for extended periods of time with
little need for specialized equipment beyond that typically
available in the laboratory. In addition, the cells prepared by the
present methods are preserved without being exposed to
cryopreservative reagents which may be toxic to the cells.
[0226] It will be readily apparent to one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein are obvious and may
be made without departing from the scope of the invention or any
embodiment thereof. Having now described the present invention in
detail, the same will be more clearly understood by reference to
the following examples, which are included herewith for purposes of
illustration only and are not intended to be limiting of the
invention.
EXAMPLE 1
Agglomeration of Typical Dry Powder Media (DPM)
[0227] 1. With a benchtop laboratory fluid bed apparatus (Stera-1;
Niro, Inc./Aeromatic-Fielder; Columbia, Md.): Place 100-500 g of
DPM within the chamber. Place onto apparatus and use the lever to
seal the unit.
[0228] 2. Start the airflow to fluidize (levitate) the DPM. Since
traditional DPM is of relatively fine particle size, setting 4-6
will be needed. Turn on the vacuum device to catch fine DPM
particles, passing through the upper filters. Make sure that the
fluidized powder is approximately central within the chamber with
respect to the lower mesh screen and the upper filters.
[0229] 3. Start the injection device (spray unit) by first plugging
in the compressed air line and then by starting the pump which is
connected to a water source. The goal is to admit .about.6 ml of
water per minute (the flow rate for any given pump based upon RPM
and tubing diameter must be known). In order to prevent clumping of
DPM, alternatively add water for .about.1 minute and then stop for
.about.1 minute, allowing drying to occur in the chamber.
[0230] 4. If filters become coated with DPM during the run so that
blowback does not dislodge powder, turn fan speed down to setting
2-3 until all filters have been blown clear. Then increase running
fan speed to previous level.
[0231] 5. Agglomeration will be complete when .about.35 ml of water
has been added for each 500 g of DPM. This volume will vary
depending upon the DPM formulation. A downward flow of relatively
large agglomerated granules will be seen in the chamber (bottom)
toward the end of the run. Visibly larger particles and absence of
fine dust indicates that the process is complete.
[0232] 6. Allow agglomerated DPM to dry thoroughly for 5-7
minutes.
[0233] 7. At end of run, blow off filters 4 times.
[0234] 8. Turn unit off, disconnect water tube and collect
agglomerated DPM into an airtight container.
[0235] These approaches should be adjusted when using a
process-scale or production-scale fluid bed apparatus. For example,
when the MP-1 (Niro, Inc./Aeromatic-Fielder; Columbia, Md.)
apparatus is used, the following protocol has yielded satisfactory
results:
[0236] 1. Seal unit (inflate gaskets).
[0237] 2. Start fan for pre-heat.
[0238] 3. Stop fan when inlet air temperature equals set point.
[0239] 4. Deflate gaskets, load material, inflate gaskets.
[0240] Steps 5-8 should all be accomplished within one minute:
[0241] 5. Start batch.
[0242] 6. Start fan, and turn on filter cleaning. [0243] 7. Set
nozzle atomizing air pressure % output (check nozzle for vacuum).
[0244] 8. Connect liquid feed line. [0245] 9. Start pump on screen
and at pump. [0246] 10. Reset batch time. [0247] 11. Spray all
liquid at set rate (26 g/min). Use 250 ml water for 2 kg powder.
[0248] 12. Stop pump at pump and on screen when all liquid is
added. [0249] 13. Reduce airflow to drying value (for example from
100 to 60). [0250] 14. When product reaches desired temperature
(.about.40.degree. C.), go to "initial set up" screen and set
"batch duration" for a value of 2-3 minutes greater than the
present "batch time". [0251] 15. Stop batch. [0252] 16. Deflate
gaskets.
[0253] Typical instrument settings (for bench-, process- and
production-scale apparatuses):
[0254] Drying temperature: 60-65.degree. C.
[0255] Outlet air temperature: .about.33.degree. C.
[0256] Blow out pressure: 5 bar
[0257] Atomizing pressure: 1.5-2.0 bar
[0258] Blow back dwell: 1 after spraying, 2 while spraying
[0259] Capacity of fan: 5 at start of run, 6 after agglomeration is
evident
[0260] Magnahelics: Filter resistance 150-250, Resistance of
perforated control plate .about.50, Air volume: less than 50.
EXAMPLE 2
Addition of Sodium Bicarbonate as an Integral Part of DPM
[0261] As noted above, sodium bicarbonate is not typically added to
DPM during manufacturing by ball-milling or lyophilization, due to
potential off-gassing and buffering capacity complications
encountered upon storage of the powdered media. This standard
production process thus necessitates the addition of sodium
bicarbonate, and pH adjustment, upon reconstitution of the media.
With the present methods, however, these additional steps may be
obviated by adding the sodium bicarbonate (or any buffering salt)
directly to the powdered medium during manufacturing.
[0262] There are two ways of including sodium bicarbonate (or any
buffering salt) within the DPM: (a) via the injection device and
(b) as part of the DPM.
[0263] (a) Injection Device
[0264] Because of the solubility of sodium bicarbonate and the
amounts that generally need to be added to a typical mammalian cell
culture medium, fairly large volumes of liquid would need to be
injected into the powder (significantly greater than the 35 ml of
water mentioned above). This is still possible and in fact may be
preferable if adding another component that similarly requires a
relatively large volume of liquid in order to be added to the DPM,
as is the case with serum for example. In this case, care must be
taken to sequentially add liquid, let dry etc. a number of times to
insure that the DPM does not become clumped within the device.
Using the 6 ml per minute for .about.1 minute and then allowing
drying for another 2 minutes is about right.
[0265] The amount of liquid to add is determined as follows:
Prepare sodium bicarbonate at 75 g/L in water. Example: 250 g of
DPM in the chamber to be agglomerated. Assume 10.0 g of DPM is
required for 1 L of 1.times. liquid medium. Therefore, 250 g
represents 25 L of 1.times. liquid medium. For each L of liquid,
assume (for example) a requirement of 2 g of sodium bicarbonate.
This means that 50 g of bicarbonate is needed. Now, since the
bicarbonate solution is at 75 g/L, then 0.67 L of bicarbonate
solution must be added to the 250 g of DPM.
[0266] The sodium bicarbonate solution would be added similarly to
the process for "agglomeration of a typical DPM" above except that
a longer drying time between cycles is needed since the pH of the
sodium bicarbonate solution is .about.8.00 which can degrade media
components. It is important that the powder never become "soaked"
by addition of bicarbonate solution too rapidly without allowing
sufficient time for thorough drying of the bicarbonate powder
between cycles. Also, longer fluid drying times are required since
it is important to have as low a final moisture content as possible
since moisture would result in liberation of carbon dioxide gas
resulting in loss of buffering capacity and "pillow" formation if
powder is in a foil packet.
[0267] (b) As Part of the DPM
[0268] Sodium bicarbonate can be milled into the DPM in a similar
fashion as for other media components prior to fluid bed treatment.
However, in the milling process, the bicarbonate should be added as
the final component. All of the other media components should be
milled as usual and then the mill stopped and the bicarbonate added
last, with further milling to reach proper sized particles. It is
important that all post-milling processing (placement into
containers, etc.) be done in a humidity-controlled environment set
as low as operationally possible (.about.20-40%. Fluid bed
processing should then be performed as soon as possible after
milling. (If not processed the same day, DPM must be double wrapped
and placed within a sealed container with moisture absorbents.)
[0269] The fluid bed process itself is done similarly to the
example given above (with use of 35 ml per 500 g of DPM) except
that drying times after water injection (.about.6 ml/min) should
again be extended: 1 min of injection of water and 2 minutes drying
cycles. It will be noted that the color of the DPM will be deep
red-light purple due to presence of phenol red. Since the DPM has
essentially no moisture content, this does not represent a
degradative situation, and is why fluid bed processing is
essential.
EXAMPLE 3
DPM that Includes Buffering Salts (e.g., Sodium Bicarbonate) and is
Formulated so that pH of Reconstituted (1.times.) Medium is
Automatically of Desired pH with No User Efforts--Spraying of Acid
or Base Technique
[0270] As noted above, all commercially available mammalian cell
culture powdered media require addition of one or more buffer salts
(e.g., sodium bicarbonate) when preparing 1.times. liquid, and then
adjustment of pH, so that the solution will be at proper pH. The
present methods, however, can be used to obviate both the addition
of sodium bicarbonate (as described above in Example 2) and the
need for pH adjustment. In this aspect of the invention, fluid bed
technology is used to introduce acid or base (depending on the
need) to a dry powder medium comprising one or more buffering
salts. In accordance with this aspect of the invention, any
buffering salts or combinations thereof, and any acid or base, may
be used depending upon the desired pH and buffering capacity in the
ultimately reconstituted cell culture medium.
[0271] If sodium bicarbonate is added directly to the DPM as a
powder, it is possible for the end user to simply add water and mix
to yield a solution already containing bicarbonate (see above) and
of proper pH. It is necessary first to determine how much of a pH
adjustment is required. (1) Place 1 L of water in a beaker. Add DPM
to the liquid and mix. (Amount to add/L is given by the
specifications for that powder, e.g., 10 g/L, 13 g/L). In this
case, the weight of the sodium bicarbonate must also be considered
in determining how much to add per liter. (2) After the powder has
dissolved, add 5N HCl to adjust the solution to the desired pH.
Record the amount. (3) Convert this number to amount of 1N HCl.
Calculate how much 1N HCl is needed for adjustment of the total
powder to be agglomerated. (Example: 5 ml of 1N HCl is needed to
adjust 1 L of 1.times. medium A to pH 7.2 from the unadjusted pH of
7.9. That 1 L of 1 .times. medium represents, for example, 13.0 g
of DPM. Therefore, for each 13.0 g of DPM, 5 ml of 1N HCl is
needed. If we want to adjust pH of 250 g of DPM, then 250 divided
by 13.0=19.2.times.5 ml or 96 ml of 1N HCl is needed to be added to
the powder to make it automatically pH-adjusted.
[0272] This 1N HCl must now be added to the DPM. The best way for
that is to use the injection device, adding 1N HCl instead of
water. In general, the protocol is similar to the above with the
following exceptions: (1) the 1N HCl must be added slowly to the
media which contains sodium bicarbonate. If it is added too
quickly, carbon dioxide may be driven off, resulting in suboptimal
buffering capacity. Because of the volume of 1N HCl generally
required, several 1 minute on, 2 minute off cycles are needed. A
dry powder state must be obtained at the end of each cycle so that
a dynamic system exists where DPM has characteristics of a fluid
process but in reality is a dried powder. (Amazingly, as HCl is
added to the powder, the bulk color changes from dark reddish
purple to light yellow-orange color even though the powder remains
essentially dry at all times due to the continual evaporation
within the system). Since the total amount of HCl has been
calculated to yield an essentially neutral pH, the powder is never
really exposed to "acid" conditions as long as the fluid bed is
properly adjusted (see above; position of the powder particles
within the chamber during operation). It is important to make sure
that all of the powder is moving through the system (i.e., being
lifted, agglomerated and settled continuously) and having no "dead"
zones within the chamber.
[0273] Once the powder is collected after the run, it can be added
to water and reconstituted at any time as long as it has been kept
in proper "dry" packaging and location. No adjustment of pH is
needed. Thus, the invention provides an automatic pH-adjusting dry
powdered medium, where the pH of the liquid medium made by
reconstituting the dry powdered medium requires no adjustment of
pH.
EXAMPLE 4
Inclusion of Large Molecular Weight Supplements Such as Serum,
Albumin, Hy-Soy, etc., within the DPM Itself
[0274] Heretofore, dried powder media containing serum have not
been commercially available. Using the present methods (via fluid
bed and spray-drying technologies), we have succeeded in adding
serum to a powder in a manner where functionality (cell culture) is
maintained.
[0275] The injection device of the fluid bed apparatus is able to
form a mist with serum, and concentrated albumin. We attempted to
see if serum added to the DPM and dried in this manner would be
functional.
[0276] Procedure for addition of serum: (1) Determine the weight of
standard DPM to be agglomerated. (2) From this, based upon the g/L
for the particular powder, calculate the volume of 1 .times. medium
that the g of powder will make. (3) Calculate the volume of serum
that would be needed at a given percentage level of supplementation
(e.g., 100 g of powder to be used in 10 g/L yields 10 L-equivalents
of powder). At 5% serum supplementation, 500 ml of serum would be
required to be added by the injection device.
[0277] Protocol for addition of the serum: Serum and albumin are
very viscous. The nozzle spray pattern must be checked for droplet
size and pattern. With the sample tube in the solution to be added
to the powder, test spray against a cardboard or other backdrop.
Check for uniformity and small droplet size. If not a "mist,"
increase atomizing pressure by 0.5 bar and test again. Do this
until sufficient pressure results in a fine mist pattern.
[0278] For use in cell culture applications, it is necessary to
know the weight/ml of serum-DPM to be used per L of 1.times.
medium. To do this, accurately weigh vials or test tubes that will
hold the serum during drying. Place a constant (known) quantity of
serum into each of the vials. Then place vials into a Speed Vac or
lyophilizer. Remove water until dryness. Then weigh the vials
again, this time containing lyophilized serum. Calculate the weight
of serum and express as per ml of original volume. The weight of
agglomerated DPM with serum to use per L will then be the standard
DPM "use" weight plus the weight of the serum at a given level.
[0279] For example, assume that Medium A (DPM) is to be used at 10
g/l. Serum supplementation is to be at 5% v/v. This means that in
addition to the weight of the standard DPM, the weight of the serum
would equal 5%=50 ml to add per L of medium. Assume that serum
powder weighs 0.06 g/ml. Then the weight of the powdered
serum=50.times.0.06 g/L=3 g. Therefore, the weight of
serum-containing DPM that would be added to 1 L of water is the
weight of serum powder (3 g) plus the weight of the standard DPM
(10 g) per liter=13 g/L.
EXAMPLE 5
Reducing or Eliminating Milling Techniques (High Energy Input
System That Break Components down to Micron-sized Particles) When
Manufacturing a DPM
[0280] As noted above, dry powdered medium typically is
manufactured via the milling process, which is laborious and has a
number of problems. The methods of the present invention provide
for the production of a dry powdered medium using fluid bed
technology, which overcomes these labor and technical
constraints.
[0281] A. Blending First in External Device, then Fluid Bed
Treatment
[0282] Normally milled DPM is blended with sodium bicarbonate
(directly as received from the supplier, additional ball milling
not needed). [RPM 1640 with sodium bicarbonate at 2
g/L-equivalents]. This mixture is blended for 20 minutes. The
powder is then placed within the fluid bed chamber and fluidized as
above for bicarbonate-containing media or bicarbonate-containing
media with automatic pH control.
[0283] B. Blending Directly in Fluid Bed Chamber, then
Agglomeration
[0284] Sodium bicarbonate is placed into the chamber directly with
the milled DPM and blended (mixed) for a brief period of time, to
be followed with agglomeration. This eliminates blending in a
separate unit.
[0285] C. Total Elimination of the Ball-milling Process
[0286] Either all of the DPM chemicals are added directly to the
fluid bed chamber and mixed preliminarily followed by agglomeration
or, more likely, some of the coarser, "stickier", etc. chemicals
are given a brief grinding treatment in a rotary grinder and then
placed within the fluid bed for blending and final
agglomeration.
EXAMPLE 6
A Method for Having All of the Above Characteristics Within this
Same DPM
[0287] We have combined addition of "off the shelf" sodium
bicarbonate with milled DPM and automatic pH control. We have also
combined serum with DPM.
[0288] To combine serum with DPM containing sodium bicarbonate with
automatic pH control, one protocol is to:
[0289] 1. Add sodium bicarbonate (powder, from supplier) to DPM
(milled or ground).
[0290] 2. Blend ingredients (mix, either external unit or fluid
bed).
[0291] 3. In a separate vessel, reconstitute 1 L of the DPM
(containing bicarbonate) with water (1.times.) and determine the
amount of 1N HCl, or 1N NaOH that is required to adjust the pH of
the solution to 7.5. On a liter basis, knowing the mass of powder
to be agglomerated (and thus the L-equivalents), calculate the
amount of 1N HCl or 1N NaOH for the total powder to be agglomerated
at the above-calculated amount. Add this amount via fluid bed
device (injection nozzle). (Although DPM is not "liquid," it is
important to have a powder as close to neutrality as possible but
not of such an acid pH that bicarbonate would be liberated when
adding serum, since moisture is involved in the process. At pH 7.6
or higher, a concentrated solution of sodium bicarbonate will not
evolve CO.sub.2 gas, but at lower pH gas will be given off.)
[0292] 4. Addition of serum (extended agglomeration), based upon
percentage supplementation and g to be agglomerated.
[0293] 5. Using the same 1 L of 1.times. liquid from (3) above,
determine the amount of 1N HCl or 1N NaOH needed to adjust the pH
to the desired pH (e.g., 7.2). Using this information, calculate
the amount to be used for the weight of powder that has been
agglomerated with serum (knowing g/L specifications). Add this
amount via fluid device (injection nozzle).
[0294] 6. Gamma irradiation is used to sterilize the powdered
media.
[0295] In a similar method, a serum-containing DPM may be produced
by combining a particular amount of DPM with a particular amount of
powdered serum (prepared, e.g., by spray-drying as described in
Example 8 below) and then agglomerating the mixture. For example,
for preparation of medium containing 10% powdered FBS, 55.5 g
powdered FBS may be added to 500 g of powdered culture medium and
the powders mixed well by agitation. This mixture may then be
water-agglomerated as described above, and will yield, upon
reconstitution, a culture medium containing 10% FBS which may be
auto-pH-adjusting.
EXAMPLE 7
Production of 100% Serum Powder by Fluid Bed Processing (To
Simulate Spray-Drying)
Methodology
[0296] 1) We used the benchtop laboratory fluid bed apparatus
(Strea-1). For production of powdered serum, nothing is placed
within the chamber. The lever is used to seal the unit.
[0297] 2) Serum was added by way of the injection device (spray
unit). As the serum was added into the chamber, the air flow was
increased enough and the flow of serum slowed enough that
evaporation of water occurred and the serum was dried sufficiently
so that powder formed instantly within the chamber. No moist or
fluid coating existed anywhere within the chamber.
[0298] 3) Pump speed was set to allow for .about.ml/minute into the
chamber.
[0299] 4) Airflow speed was set to a setting of .about.8-9.
[0300] 5) To clean filters intermittently, fan speed was reduced to
.about.2-3. This was done routinely every 5-10 minutes. (The 8-9
airflow setting is so high that the filters will not blow off the
powder and clean themselves).
[0301] 6) After one round of filter blow-off, fan speed was
increased to previous levels and the pump turned on. (Once these
parameters were set, the pump was run continuously except when
cleaning the filters as indicated).
[0302] 7) After all of the serum liquid had been added into the
agglomerator, final drying was performed over five minutes.
[0303] 8) The filters were then blown off to collect as much powder
as possible, and the machine shut off and product removed. Powdered
serum was placed into an air-tight container and protected from
light.
Typical Instrument Settings
[0304] Drying temperature: 60-65.degree. C.
[0305] Outlet air temperature: -33.degree. C.
[0306] Blow out pressure: 5 bar
[0307] Atomizing pressure: 2.0-2.5 bar
[0308] Blow back dwell: 2, in between spraying
[0309] Capacity of fan: 8-9 throughout run
[0310] Magnahelics: Filter resistance--150-250, Resistance of
perforated control plate--.about.50, Air volume--less than 50.
[0311] To determine if agglomeration of the FBS affected the
protein structure or distribution, samples of agglomerated FBS and
liquid FBS were run on SDS-PAGE, stained for protein and scanned
densitometrically. As shown in FIG. 1, agglomerated FBS prepared
according to the present methods (FIG. 1A) demonstrated a nearly
identical protein profile to that observed with liquid FBS (FIG.
1B). These results indicate that the controlled production of dry
powdered FBS by the present methods does not substantially affect
the structure or distribution of the major components of the
serum.
[0312] To determine if agglomeration of the FBS affected its
ability to support cell growth and passage, SP2/0 cells were plated
into DMEM containing either 2% agglomerated ("dry") FBS or 2%
liquid FBS and growth rates and passage recovery examined. As shown
in FIG. 2A, cells plated into media containing agglomerated FBS
demonstrated similar growth kinetics as did cells plated into media
containing liquid FBS. Similarly, cells in media containing
agglomerated FBS recovered from passage with practically identical
growth rates as cells in media containing liquid FBS (FIG. 2B).
Together, these results indicate that the agglomerated FBS of the
present invention performs approximately equivalently to liquid FBS
in supporting growth and passage of cultured cells.
EXAMPLE 8
Production of 100% Serum Powder by Spray-Drying
[0313] As an alternative to fluid bed processing, the feasibility
of producing dry powdered serum by spray-drying technology was
examined. A three foot diameter laboratory spray drier (Mobile
Minor Spray Drier; NIRO, Columbia, Md.) was used to prepare the
powdered serum. Liquid FBS was aspirated into the spray-dryer and
atomized through a Schlick 940 nozzle located in the middle of the
air dispenser, and the drying air was introduced into the atomizer
through the top air dispenser of the apparatus. Spray drying was
conducted under the following conditions: inlet air
temperature=200.degree. C.; outlet air temperature=70.degree. C.,
atomizing air pressure for the nozzle=2.0 bar; air flow=80.0
kg/hour; spray rate=65 g/minute. During development of these
methods, an initial outlet air temperature of 60.degree. C. was
used; however, this temperature was found to be too low, and the
spray rate was adjusted back to a level to achieve an outlet
temperature of about 70.degree. C. which was found to be optimal.
Following spray-drying, powdered serum was collected at the cyclone
of the apparatus, and process air was filtered through an exhaust
filter prior to recirculation within the apparatus.
[0314] Following production, the powdered serum was characterized
with respect to its physical properties, compared to liquid FBS
from the same source lot. Samples taken from different stages of
the production lot (samples "A" and "B") were reconstituted at a
concentration of 60.44 g/L in endotoxin-free distilled water
(Invitrogen Corporation, Carlsbad, Calif.), and were examined for
endotoxin levels using a Limulus Amoebocyte Lysate test (Invitrogen
Corporation, Carlsbad, Calif.), for hemoglobin levels (by
spectrophotometrically measuring absorbance at 525 nm), and by
UVNis spectrophotometry. Results are shown in Table 1, and in FIGS.
3A and 3B. TABLE-US-00001 TABLE 1 Physical Characterization of
Powdered Serum. Material Tested Endotoxin Level (EU/ml) Hemoglobin
(mg/100 ml) Powdered FBS, 0.6 7.7 Sample "A" Powdered FBS, <0.3
7.7 Sample "B" Liquid FBS <0.3 7.2 (control)
[0315] As seen in Table 1, powdered FBS demonstrated endotoxin and
hemoglobin levels similar to those of the liquid FBS that served as
the source material for production of the powdered FBS. Moreover,
samples taken from different stages of the production process
demonstrated nearly identical endotoxin and hemoglobin levels,
indicating that the present methods result in the production of
material with approximately uniform physical consistency across the
production lot. When samples of powdered and liquid FBS were
examined by UV/visible spectrophotometry (FIG. 3), the trace
observed for powdered FBS (FIG. 3A) was indistinguishable from that
obtained for the source liquid FBS (FIG. 3B). Together, these
results indicate that serum powder prepared by the present
spray-drying methods have nearly identical physical characteristics
as those of liquid sera from which the powders are prepared. Taken
together with those of Example 7 above (see, e.g., FIG. 1), these
results demonstrate that the methods provided by the present
invention result in the production of powdered sera with physical
characteristics that are unaltered from those of the source liquid
sera.
EXAMPLE 9
Production of Automatically pH-Adjusted Powdered Culture Media
[0316] One reason that sodium bicarbonate is never included in
powdered media is that any moisture, even that in the air, may
result in an acidic condition within the pouch that will result in
the liberation of CO.sub.2 gas. The pouches will become swollen and
produce what have been called "pillows." With fluid bed processing,
the humidity within the apparatus is reduced essentially to
negligible levels prior to the end of the process. We have made
RPMI-1640 powdered media containing sodium bicarbonate and have not
seen evidence of "pillow" formation.
[0317] In order to make a pH-adjusted powdered media, it is
necessary to add the pH-adjusting chemical (usually HCl or NaOH) to
the powder to bring the pH to about 7.0-7.4 upon addition to water.
Once sodium bicarbonate is added to the powder, many powdered media
reconstitute in water on the basic side of neutrality and need HCl
addition. Adding HCl to a powder containing sodium bicarbonate
would be expected to be problematic. However, since the added
liquid (5N HCl in this case) never results in a moistened or
"liquid" state inside the fluid bed apparatus, the sodium
bicarbonate does not give off CO.sub.2 gas and fully retains its
buffering capacity. This has been examined in the present studies
by pH-titering experiments: equal amounts of acid, in two separate
experiments (FIGS. 4A and 4B) were found to reduce the pH of
agglomerated media and automatic pH-adjusted agglomerated media by
an identical amount as that for a standard medium with sodium
bicarbonate added to the liquid at the time of reconstitution.
These results indicate that both agglomeration with subsequent
adjustment of pH, and agglomeration with adjustment of pH during
the agglomeration process, function equally well to produce
powdered culture media with significant buffering capacity.
EXAMPLE 10
Effect of Agglomeration on Dissolution Rates of Culture Media
[0318] To examine the effect of agglomeration of culture media on
the rate of dissolution of the media, samples of Opti-MEM I.TM. or
DMEM were agglomerated with water or with FBS (2% only for Opti-MEM
I; 2% or 10% for DMEM). Upon reconstitution of the agglomerated
media in water, the time dissolution of the agglomerated Opti-MEM I
occurred much more quickly than did dissolution of standard
powdered Opti-MEM I (FIG. 5A); results were identical for water-
and FBS-agglomerated Opti-MEM I. Interestingly, however, while
water-agglomerated DMEM dissolved in water much more quickly than
did standard powdered DMEM, the FBS-agglomerated DMEM did not (FIG.
5B).
[0319] Due to the open structure of the agglomerated powdered media
(as opposed to traditional powdered media), capillary action brings
water into close proximity with all of the powder particles. This
prevents the appearance of powder "balls," a complication observed
upon reconstitution of most standard powdered media that leads to
longer dissolution times. In addition to more rapid dissolution,
agglomerated media demonstrated reduced dusting as well. These
results indicate that water-agglomerated culture media, and some
FBS-agglomerated culture media, are much more rapidly dissolving
and generate less dust than traditional powdered culture media.
EXAMPLE 11
Cell Growth and Subculturing in Reconstituted Agglomerated Culture
Media
[0320] Many uses of culture media require additions of large
molecular weight proteins such as serum or albumin. These molecules
may be in the form of solutions or even powder in the case of
albumin. However, in order to insure uniformity of powdered media,
these proteins are usually added not as a powder but as liquid
after reconstitution of the bulk powdered media to a liquid medium.
This presents some inconvenience since, for example, serum must be
stored in the freezer to maintain performance over time. This adds
expense and inconvenience since the serum must be added aseptically
to the media, increasing chances of contamination. If filtration is
done after addition of serum, another processing step is needed.
There would therefore be advantages to being able to provide serum
as an integral part of the powdered media.
[0321] Therefore, culture media were agglomerated with water or
with various concentrations of FBS. FBS was added to the powdered
media by injecting it into the air-suspended dry powdered media at
high evaporation rates, as generally outlined above. The level of
serum supplementation was 2% in Opti-MEM I media, and 2% or 10% in
DMEM. The growth and passage success of various cell lines in these
media were then assessed.
[0322] As shown in FIG. 6, SP2/0 cells demonstrated similar growth
rates when grown in Opti-MEM I agglomerated with either water or
with FBS (FIG. 6A), compared to cells grown under conventional
culture conditions (liquid serum added to water-reconstituted
powdered media). Similar results were observed with SP2/0 cells
cultured in water- and FBS-agglomerated DMEM supplemented with 2%
FBS (FIG. 6B), and with SP2/0 cells (FIG. 7A), AE-1 cells (FIG. 7B)
and L5.1 cells (FIG. 7C) cultured in water- and FBS-agglomerated
DMEM supplemented with 10% FBS. In addition, SP2/0 cells showed
approximately similar recovery rates from passage when cultured in
water- or agglomerated Opti-MEM I and DMEM supplemented with 2% FBS
(FIGS. 8A and 8B, respectively), as did SP2/0 cells, AE-1 cells and
L5.1 cells cultured in water- and FBS-agglomerated DMEM
supplemented with 10% FBS (FIGS. 9A, 9B and 9C, respectively) and
SP2/0 cells cultured in water-agglomerated DMEM supplemented with
5% FBS (FIG. 10). Furthermore, SP2/0 cells demonstrated identical
passage characteristics in water-agglomerated media produced in
large batches and in automatically pH-adjusting powdered DMEM
containing sodium bicarbonate as they did in standard liquid DMEM
supplemented with 5% FBS (FIG. 10).
[0323] Together, these results indicate that culture media
supplements such as animal sera (e.g., FBS) may be agglomerated
directly into culture media, and that supplementation of culture
media during the agglomeration process in this way produces a
culture medium that provides optimal support of growth and passage
of a variety of cultured cells. Furthermore, these results indicate
that the present culture media powders may be successfully produced
in large batches, including the automatically pH-adjusting media of
the invention that contain sodium bicarbonate.
EXAMPLE 12
Cell Growth in Culture Media Supplemented with Spray-Dried Serum
Powder
[0324] As a corollary to the experiments shown in Example 7, AE-1
cells and SP2/0 cells were plated into DMEM containing either 2% or
10% spray-dried FBS prepared as described in Example 8, or
containing 2% or 10% liquid FBS, and growth rates and passage
recovery of the cells were examined. Cells were inoculated into
triplicate 25 cm.sup.2 flasks at a density of 1.times.10.sup.5
cells/ml in 10 ml of media. Viable cell density was determined on
days 3-7, and each cell line was tested twice. Results are shown in
FIGS. 11-13.
[0325] As shown in FIG. 11, AE-1 cells cultured in media containing
powdered FBS demonstrated similar growth kinetics to those cells
cultured in media containing standard liquid FBS. As expected, the
cells demonstrated more rapid growth to a higher density in culture
media containing 10% FBS than in media containing 2% FBS, and
demonstrated peak growth by about day four. Similar kinetics were
observed for two separate experiments (FIGS. 11A and 11B),
indicating that these results were reproducible. Analogous results
were obtained in two experiments in which the growth rates of SP2/0
cells were measured in media containing powdered or liquid FBS
(FIGS. 12A and 12B). In addition, AE-1 cells cultured in media
containing 5% powdered FBS recovered from passage with identical
growth rates as cells in media containing liquid FBS (FIG. 13).
[0326] These results indicate that the powdered FBS prepared by the
spray-drying methods of the present invention performs
approximately equivalently to liquid FBS in supporting growth and
passage of cultured cells. Together with those from Examples 7 and
8, these results indicate that the methods of the present invention
may be used to produce powdered FBS, by fluid bed or spray-drying
technologies, that demonstrates nearly identical physical and
performance characteristics as those of liquid FBS.
EXAMPLE 13
Effect of Irradiation on Performance of Agglomerated Media
[0327] Recently, concerns have been raised about the biological
purity of media and media components (including supplements) used
for bioproduction, particularly in the biotechnology industry.
Gamma irradiation is a sterilization process that is known to work
well with certain liquids and powders that are not typically
amenable to sterilization by heat or toxic gas exposure. Therefore,
samples of water- or FBS-agglomerated culture media were .gamma.
irradiated with a cobalt source at 25 kGy for up to several days,
and the growth rates of various cell types examined.
[0328] In one set of experiments, SP2/0 cells were inoculated into
various media at 1.times.10.sup.5 cells/ml and cultured at
37.degree. C. At various intervals, samples were obtained
aseptically and cell counts determined by Coulter counting and
viability determined by trypan blue exclusion. Media were prepared
by dissolving sufficient powdered media to make a 1.times. solution
in 1L of water, stirring and filtering through a 0.22 .mu.m filter.
Results are shown in the graph in FIG. 14. Those conditions on the
graph that state "pwdr FBS" on the graph refer to the addition of
powdered FBS (prepared as in Examples 7 or 8 above) to the
reconstituted 1.times. medium prepared from either standard
powdered media or from agglomerated media (irradiated or
non-irradiated). Those conditions on the graph that state "Irradia.
agglom. DMEM +FBS" refer to use of the fluid bed to make the
agglomerated media by spraying FBS into the powdered media
(standard or agglomerated) to make an FBS-agglomerated media.
[0329] As shown in FIG. 14, .gamma. irradiation of standard
powdered basal media and agglomerated basal media did not
deleteriously affect the ability of these media to support SP2/0
cell growth. In addition, while irradiation did negatively impact
powdered media containing powdered FBS, and powdered FBS itself,
this effect diminished with increasing serum concentration.
[0330] To more broadly examine these .gamma. irradiation effects,
samples of VERO cells were inoculated into VP-SFM.TM. that had been
conventionally reconstituted or agglomerated as above. To the
powdered media in the agglomeration chamber, however, epidermal
growth factor (EGF) and ferric citrate chelate, traditional
supplements for this media, were added via the spray nozzle during
agglomeration. Media were then used directly or were .gamma.
irradiated as described above. Cells were inoculated at
3.times.10.sup.5 cells/flask into T-25 flasks and incubated at
37.degree. C. Cell counts and viability were performed as described
above, with results shown in FIG. 15.
[0331] As seen in FIG. 15, VERO cells demonstrated approximately
equivalent growth and passage success when cultured in agglomerated
media that had been .gamma.-irradiated as in agglomerated media
that had not been .gamma.-irradiated. Furthermore, irradiation of
the media had no effect on the low-level culture supplements EGF
and ferric citrate chelate that were present in the media.
[0332] These results indicate that .gamma. irradiation may be used
as a sterilization technique in the preparation of many bulk
agglomerated culture media, including those containing serum, EGF
or other supplements, by the present methods.
EXAMPLE 14
Effect of Irradiation on Performance of Powdered Media
Supplements
[0333] To demonstrate the efficacy of the present methods in
producing sterile media supplements, lyophilized human
holo-transferrin was irradiated by exposure to a cobalt .gamma.
source at 25 kGy for about 3 days at -70.degree. C. or at room
temperature. 293 cells were then cultured in media that were
supplemented with irradiated transferrin or with control
transferrin that had not been irradiated (stored at -70.degree. C.
or at room temperature), and cell growth compared to that of
standard transferrin-containing culture media or media that
contained no transferrin.
[0334] Mid-log phase 293 cells that were growing in serum-free 293
medium (293 SFM) were harvested, washed once at 200 .times. g for 5
minutes and resuspended in transferrin-free 293 SFM for counting
and viability determination. Cells were plated into triplicate 125
ml Ehrlenmeyer flasks at a density of 3.times.10.sup.5 cells/ml in
a volume of 20 ml in 293 SFM (positive control), transferrin-free
293 SFM (negative control), in 293 SFM containing non-irradiated
transferrin stored at -70.degree. C. or at room temperature, or in
293 SFM containing irradiated transferrin prepared as described
above. Flasks were placed into a rotary shaker set at about 125
rpm, in a 37.degree. C. incubator equilibrated with an atmosphere
of 8% CO.sub.2/92% air. Daily cell counts were determined using a
Coulter particle counter and viabilities were determined by trypan
blue exclusion according to standard procedures. When the cells
reached a density of about 1.2 to 1.7.times.10.sup.6 per flask, the
contents of one of the flasks of each sample were harvested,
centrifuged, resuspended into fresh medium and passaged into three
new flasks. Cell counts and viabilities of the previous and next
passages were then performed as described above. Four consecutive
passages of cells incubated under the above conditions were
tested.
[0335] As shown in FIGS. 16A-16D, cells cultured in media
containing transferrin that was .gamma. irradiated at either
-70.degree. C. or at room temperature demonstrated nearly identical
growth kinetics and survival in the first passage (FIG. 16A),
second passage (FIG. 16B), third passage (FIG. 16C) and fourth
passage (FIG. 16D) as did cells cultured in standard 293 SFM or in
293 SFM containing transferrin that had not been .gamma.
irradiated. Cells cultured in transferrin-free media, however,
survived well during the first passage (FIG. 16A) but stopped
growing and demonstrated a significant loss in viability upon
subculturing (FIG. 16B).
[0336] These results demonstrate that .gamma. irradiation may be
used as a sterilization technique in the preparation of bulk
powdered culture media supplements, such as transferrin, in the
methods of the present invention. Furthermore, these data indicate
that culture media supplements such as transferrin may be .gamma.
irradiated at room temperature without significant loss of
activity.
EXAMPLE 15
Effect of Irradiation on Biochemical Characteristics of Powdered
Sera
[0337] To further determine the impact of .gamma. irradiation on
sera, samples of spray-dried powder FBS were irradiated at 25 kGy
at -70.degree. C. or at room temperature (RT), and were analyzed
commercially for the concentrations of various biochemical
constitutents in the sera. As controls, samples of non-irradiated
spray-dried FBS and liquid FBS were also analyzed. Results are
shown in Table 2. TABLE-US-00002 TABLE 2 Chemical Analysis of
Spray-Dried FBS Dried Dried Non-ir- FBS, FBS, radiated Irr. @ Irr.
Dried Liquid Reference Constituent -70.degree. C. @RT FBS FBS Units
Range Sodium 139 137 139 140 mM 136-144 Potassium 13.2 13.2 13.0
13.2 mM 3.6-5.2 Chloride 98 97 98 100 mM 98-108 Uric Acid 1.6 1.3
1.7 1.9 mg/dL 2.2-8.3 Phosphorus 10.1 10.1 9.6 10.2 mg/dL 2.2-4.6
Calcium 14.9 14.8 14.8 14.5 mg/dL 8.6-10.2 Ionizable >5.5
>5.5 >5.5 >5.5 mg/dL 3.8-4.5 Calcium Magnesium 2.77 2.76
2.75 2.76 meg/L 1.4-2.0 Alkaline 57 47 68 269 U/L 31-142
Phosphatase Gamma GT 3 5 <5 5 U/L 1-60 (GGTP) AST 7 5 5 33 U/L
1-47 (SGOT) ALT (SGPT) 5 <5 <5 7 U/L 1-54 LD 56 <50 50 510
U/L 110-250 Total 0.19 0.24 0.22 0.13 mg/dL 0.2-1.4 Bilirubin
Direct 0.04 0.07 0.07 0.04 mg/dL 0.0-0.3 Bilirubin Glucose 67 38 39
88 mg/dL 65-125 BUN 15 15 15 15 mg/dL 6-23 Creatinine 2.98 3.08 3.1
2.77 mg/dL 0.1-1.7 BUN/ 5.0 4.9 4.8 5.4 -- 7.0-20.0 Creatine Ratio
Total Protein 3.6 3.6 3.5 3.7 gm/dL 6.4-8.1 Albumin 2.7 2.7 2.8 2.8
gm/dL 3.7-5.1 Globulin 0.9 0.9 0.7 0.9 gm/dL 2.1-3.6 Albumin/ 3.0
3.0 4.0 3.1 -- 1.1-2.3 Globulin Ratio Cholesterol 30 30 32 30 mg/dL
<200 HDL 28 30 30 27 mg/dL 39-90 Cholesterol Chol/HDL 1.07 1.00
1.07 1.11 -- <4.5 Ratio Triglycerides 72 74 72 73 mg/dL 30-200
Iron 213 217 214 186 meg/dL 40-175 Plasma Hb 13.3 11.5 13.7 22.6
mg/dL 3.4-20.5
[0338] These results indicate that the .gamma. irradiation process
did not significantly affect the concentrations of most of the
biochemical constituents of FBS. These results also indicate that
upon spray-drying, several of the components of FBS (alkaline
phosphatase, AST, and LD, and possibly glucose) undergo a
significant reduction in concentration compared to their
concentrations in the starting liquid FBS.
EXAMPLE 16
Effects of Irradiation on Performance of Powdered Sera
[0339] To examine the impact of .gamma. irradiation on the ability
of dried powder sera to support cell growth, samples of spray-dried
FBS irradiated under various conditions were used to supplement
culture media, and adherent and suspension cells were grown for up
to three passages in these media. As model suspension cells, the
hybridoma lines SP2/0 and AE-1 were used, while VERO and BHK
cultures were used as typical adherent cells. Cells were cultured
in media containing test sera or control sera (spray-dried but not
irradiated) for up to three passages according to the general
procedures outlined in Example 14 above. At each passage point,
cells were harvested and subcultured, while an aliquot was counted
as above for viable cells/ml. Results at each point were expressed
as a percentage of the viable cell count obtained in media
supplemented with liquid FBS, and are shown in FIGS. 17A, 17B, 17C
and 17D.
[0340] Several conclusions may be drawn from the results of these
studies. First, .gamma. irradiation of FBS does not appear to
reduce the ability of spray-dried FBS to support the growth of
suspension and adherent cells (compare the irradiated data sets to
the non-irradiated data set in each figure). In fact, BHK cells
(FIG. 1 7D) actually grew better in media containing powdered FBS
that had been irradiated at -70.degree. C. than they did in
non-irradiated sera. Second, sera irradiated at -70.degree. C.
appear to perform better than those irradiated at room temperature
in their ability to support cell growth, except perhaps for VERO
cells (FIG. 17C). Finally, the results of these studies were very
cell type-specific: suspension cells (FIGS. 17A and 17B) grew
better in spray-dried FBS, irradiated and non-irradiated, than did
adherent cells (FIGS. 17C and 17D); and among adherent cells, BHK
cells (FIG. 17D) grew better in spray-dried FBS than did VERO cells
(FIG. 17C).
[0341] These results demonstrate that .gamma. irradiation may be
used as a sterilization technique in the preparation of bulk
powdered sera, such as FBS, in the methods of the present
invention. Furthermore, unlike those reported for transferrin in
Example 14 above, these data suggest that the optimal temperature
for irradiation of sera, in order to maintain the ability of the
sera to support cell growth, is likely to be below room
temperature.
EXAMPLE 17
Production of Automatically pH-Adjusted Powdered Culture Media by
Phosphate Balancing
[0342] As noted above, typical commercially available mammalian
cell culture powdered media require addition of one or more buffer
salts (e.g., sodium bicarbonate) when preparing a 1.times. liquid,
followed by adjustment of pH, so that the solution will be at
proper pH for use. The methods of the present invention, however,
can be used to obviate both the post-reconstitution addition of
sodium bicarbonate (as described above in Example 2) and the need
for pH adjustment. In this aspect of the invention, fluid bed
technology may be used to introduce acid or base (depending on the
need) to a dry powder medium comprising one or more buffering
salts. In accordance with this aspect of the invention, any
buffering salts or combinations thereof, and any acid or base, may
be used depending upon the desired pH and buffering capacity in the
ultimately reconstituted cell culture medium.
[0343] If sodium bicarbonate is added as a powder directly to the
dry powder medium (DPM) in accordance with the methods of the
present invention, it is possible for the end user to simply add
solvent (e.g., water) and mix to yield a solution already
containing bicarbonate (see above) and at the proper pH for
immediate use--i.e., an "auto-pH" or "automatically pH-adjusting"
culture medium of the invention. To determine how much of a pH
adjustment is required, several steps should be undertaken:
[0344] (1) Place .about.950 ml of solvent (e.g., water) in a
beaker. Add DPM (in an amount according to the manufacturer's
specifications or according to formulation specifications that are
known in the art as referred to herein) to the solvent and mix
quantum sufficit to 1L. In this case, the weight of the sodium
bicarbonate must also be considered in determining how much DPM to
add per liter. However the DPM should contain neither sodium
phosphate buffer nor HEPES buffer, which will be titrated in, but
should contain sodium bicarbonate.
[0345] (2) The next step is to determine whether monobasic or
dibasic phosphate will give the desired final pH. This depends on
the whether the adjustment ofpH needed is to a more basic level
(indicating the need for dibasic phosphate), or to a more acidic
level (indicating the need for monobasic phosphate). Sequentially
add amounts (at final concentration ranges of about 0.1 mM to about
10 mM, about 0.2 mM to about 9 mM, about 0.3 mM to about 8.5 mM,
about 0.4 mM to about 8 mM, about 0.5 mM to about 7.5 mM, about 0.6
mM to about 7 mM, and preferably about 0.7 mM to about 7 mM) of
monobasic or dibasic phosphate salts to obtain the desired pH. The
total molar amounts of sodium phosphate should remain constant, but
buffering due to either monobasic or dibasic results in similar
buffering kinetics at a given pH (see FIG. 18), because the
molecular species in solution is the same as determined by the
final (desired) pH of the solution.
[0346] (3) If the medium contains a HEPES buffer system, add the
correct molar amount of either the acid form (for more acidic pH)
or the sodium form (for more basic pH) of HEPES to arrive at the
proper (desired) final pH.
[0347] (4) The next step is to exchange the monobasic sodium
phosphate for monobasic potassium phosphate on a molar weight basis
(identical buffering characteristics result from use of either
sodium or potassium phosphate). To carry out this exchange, the
amount of monobasic sodium phosphate calculated in step 2 above is
substituted with an equal amount of potassium phosphate, which is
then used to formulate the final medium. Hence, in this aspect of
the invention, the amount of monobasic sodium phosphate calculated
in step 2 above is not actually added to the medium to adjust the
pH; instead, this amount is simply calculated, and then the
calculated amount of potassium phosphate is used in adjusting the
pH of the final medium. This is done so that subsequent off-gassing
of carbon dioxide gas is eliminated or minimized. (In order to have
the same molar ratios of potassium to sodium in the formulation as
a whole, it is necessary to back-adjust the amount of potassium
chloride in the formulation so that the final 1.times. molar
amounts are the same. This may also result in the need to adjust
with a small amount of sodium chloride to reach identical
osmolarities). When the solution is in its final form, it is dried
into a powder by either agglomeration using fluid bed technology
(as described in Example 1), spray-drying (see Example 8), or
lyophilization techniques known to those skilled in the art.
[0348] While the above are the basic manipulations recommended for
including sodium bicarbonate in powdered media, additional
considerations should also be kept in mind:
[0349] 1. Only anhydrous forms of media components should be
used
[0350] 2. If anhydrous forms are not available, consider using
"ionic replacement" (e.g., ZnSO.sub.4.7H.sub.2O or monohydrous;
also consider using ZnCl.sub.2 with sodium sulfate and reducing
stoichiometrically correct amounts of NaCl from the added amount of
NaCl as indicated in the formulations);
[0351] 3. Do not use HCl conjugates of chemicals: use free base
(ex. arginine instead of arginine HCl, corrected for true desired
weight);
[0352] 4. Monobasic sodium phosphate should not be used at all
since it can cause pillowing. Instead, monobasic potassium
phosphate (KH.sub.2PO.sub.4), which does not cause pillowing should
be used. The buffering responses of both of these chemicals is
identical. The amount of potassium added to the formulation in the
forms of other salts should correspondingly be reduced by the
amount KH.sub.2PO.sub.4 added here. Also, extra NaCl may be needed
so that the osmolarity of the formula with sodium phosphate is
equal to the osmolarity of the formula with potassium
phosphate.
[0353] 5. Dibasic sodium phosphate (Na.sub.2HPO.sub.4) does not
"pillow" and is acceptable for use in the formulation.
[0354] 6. Do not use HCl "spray in" in the fluid bed for
auto-adjust mechanism because this increases the propensity to
"pillow" with the bicarbonate. Instead, adjust the final pH of
reconstituted media by phosphate balance in the DPM.
(Na.sub.2HPO.sub.4 raises pH, while KH.sub.2PO.sub.4 reduces pH. As
indicated above use "ionic balance" results in the same ionic
composition of the 1.times. media).
[0355] 7. If amino acid(s) need to be added as a spray-in for the
fluid bed (such as cysteine for CD-CHO) because of solubility
concerns, do not reduce pH to dissolve amino acids but raise pH to
the minimum needed for solubilization (e.g., pH 10.66 for
cysteine).
[0356] 8. If a component cannot be obtained anhydrous or "ionic
replacement" does not apply, the chemical can be solubilized and
sprayed into the media via agglomeration: water in the crystal
component will not dry during agglomeration, but if the water is
released by dissolution, then it will be eliminated from the DPM by
the spraying and evaporation process.
[0357] 9. Check with an accelerated shelf life test (37.degree. C.
in a sealed pouch with equal amounts of bicarbonate for any
chemicals that appear moist or gooey such as choline chloride. One
example of an acceptable shelf life testing protocol is as follows:
[0358] (a) place 10 grams of the test compound into a mortar and
pestle; add 10 grams of NaHCO.sub.3; [0359] (b) grind the mixture
to reduce particle size and blend for about 30 seconds; [0360] (c)
add the blended mixture to a foil pack; seal the open end of the
pack; [0361] (d) place the pack into an incubator at 37.degree. C.,
and observe for "pillow" formation (i.e., swelling of the pack due
to off-gassing of the NaHCO.sub.3) over 24 hours. If gas is given
off, solubilize and add that component to the spray-in
solution.
[0362] 10. Place choline Cl in neutral solubles spray-in.
[0363] Once the powder is collected after the spray-dry or
agglomeration run, it can be reconstituted with a solvent (e.g.,
water) at any time, as long as it has been kept in proper "dry"
packaging and conditions. Examples of acceptable or "proper" dry
packaging include any packaging that retards or prevents the
penetration of water and/or water vapor through the packaging upon
storage, such as foil packaging, polyethylene bags, sealed plastic
(particularly polypropylene, polycarbonate, polystyrene,
polyethylene terephthalate (PET) and the like). Examples of proper
storage conditions include storage at about 0.degree. C. to about
25.degree. C., preferably about 2.degree. C. to about 20.degree.
C., about 2.degree. C. to about 15.degree. C., about 2.degree. C.
to about 10.degree. C., or about 2.degree. C. to about 8.degree.
C., under diminished or subdued lighting. Under such conditions,
minimum shelf life of the media of the present invention is about
one year (stored at about 2.degree. C. to about 8.degree. C.), or
about six months (at about 20.degree. C. to about 25.degree. C.
(room temperature)).
[0364] For use, the powder is simply reconstituted with an
appropriate solvent (e.g., water); no adjustment of pH is needed,
since the media are at the appropriate pH and have appropriate
buffering kinetics immediately upon reconstitution (see FIGS. 19A
and 19B). Thus, the invention provides an automatic pH-adjusting
dry powdered medium, where the pH of the liquid medium made by
reconstituting the dry powdered medium requires no adjustment of
pH.
EXAMPLE 18
Spray in of Media Components in .mu.g/ml or .mu.g/L Quantities
[0365] Chemicals such as trace elements (such as calcium, copper,
iron, magnesium, manganese, nickel, potassium, tin, and zinc,
vitamins (such as A, B.sub.1, B.sub.2, B.sub.6, B.sub.12, C, D, E,
K and H (biotin), viral inhibitors (such as protease inhibitors,
nucleoside analogues, and the like), growth factors (such as EGF,
aFGF, bFGF, HGF, IGF-1, IGF-2, and NGF), etc., may be added to
standard powdered media by first making a concentrate of the
chemicals and then spraying them into the powdered media
granulation (see U.S. patent application Ser. No. 09/023,790, filed
Feb. 13, 1998, which is incorporated herein by reference in its
entirety.) The resulting powder may then be milled (e.g., with a
Fitzmill) to a particle size in the same general size range as that
of the bulk for blending (which is required after weighing and
Fitzmilling). This portion may then be combined with the bulk
powdered medium and milled together to create a homogeneously mixed
powdered medium. Alternatively, the components of the powdered
media may be subgrouped into mixtures of compatible compounds or
components, which may then be blended immediately prior to
formulation, and concentrates of the low-level components may be
sprayed into the blend. This approach is particularly advantageous
when dealing with components that may be incompatible if they are
admixed and stored together for extended times in a powdered media
formulation (e.g., cysteine and glutamine, which will form
insoluble complexes upon storage together for extended periods of
time). (For a more detailed description of the advantages of
subgrouping culture medium components, see Example 19 below, and
commonly owned U.S. Pat. Nos. 5,474,931 and 5,681,748, the
disclosures of which are incorporated by reference herein in their
entireties). The ability to spray-in chemicals in small amounts is
especially helpful in developing media that contains components
present in small quantities and which are inconvenient to add
separately. Thus, the dry powdered medium is ready to use.
EXAMPLE 19
Subgrouping of Components to Avoid Harmful Interactions During
Agglomeration
[0366] Some of the components of a medium may be incompatible and
cause interact deleteriously to each other if they are weighed and
then held together prior to milling and agglomeration. For example,
adverse reactions have been observed when cysteine and glutamine
powders are admixed, when phosphate salts are admixed with calcium-
or magnesium ion-containing salts, when phosphate salts
(particularly monobasic forms thereof) are admixed with choline
chloride, and when glutathione is admixed with amino acids. In
addition, acidic components (e.g., acidic forms of certain buffer
salts, vitamins, and the like) may denature protein components such
as growth factors or serum. Therefore, these particular components
can be subgrouped (see commonly owned U.S. Pat. Nos. 5,474,931 and
5,681,748, which describe methods of subgrouping culture media,
supplement and buffer components, and the disclosures of which
patents are incorporated herein by reference in their entireties)
and agglomerated together as a subgroup. Specific subgroups
include, an acid soluble subgroup, a weak acid-base soluble
subgroup, a glutamine-containing subgroup, an alcohol soluble
subgroup, an alkali-soluble subgroup, and a supplement-containing
subgroup. After agglomeration of the separate subgroups, they can
then be mixed together, as described in Example 18 for spraying in
of elements in small quantities.
[0367] Alternatively, the concentrates may be sprayed directly into
already-milled bulk powder. In this approach, each subgroup is
milled (e.g., via Fitzmilling) and then placed into the fluid bed
apparatus at the same time as other subgroups, so that the
subgroups are mixed together during agglomeration; concentrates may
then be sprayed into the bulk powder sequentially, such that
individual incompatible components are only admixed for a very
short period of time prior to being agglomerated. The agglomerated
powder can then be collected and stored as described herein until
reconstitution and use, without adverse reactions occurring among
individual, otherwise incompatible, components.
[0368] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious to one of ordinary skill in
the art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are encompassed within the scope of the
appended claims.
[0369] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
* * * * *